Conditionally Active Chimeric Antigen Receptors for Modified T-Cells

ABSTRACT

This disclosure relates to a chimeric antigen receptor for binding with a target antigen. The chimeric antigen receptor comprises at least one antigen specific targeting region including a multispecific bivalent monovalent antibody evolved from a wild-type antibody or a fragment thereof and having at least one of: (a) a decrease CAB-scFv Affinity ELISA in activity in the assay at the normal physiological condition compared to the wild-type antibody or the fragment thereof, and (b) an increase in activity in the assay under the aberrant condition compared to the wild-type antibody or the fragment thereof. A method for using the chimeric antigen receptor and cytotoxic cells for cancer treatment is also provided.

FIELD OF THE DISCLOSURE

This disclosure relates to the field of protein evolution. Specifically,this disclosure relates to a method of generating a conditionally activechimeric antigen receptor from a wild type protein. The conditionallyactive chimeric antigen receptor is reversibly or irreversiblyinactivated at a wild type normal physiological condition, but is activeat an aberrant condition.

BACKGROUND OF THE DISCLOSURE

There is a considerable body of literature describing the potential forevolving proteins for a variety of characteristics, especially enzymes.For example, enzymes may be evolved to be stabilized for operation atdifferent conditions such as at an elevated temperature. In situationswhere there is an activity improvement at the elevated temperature, asubstantial portion of the improvement can be attributed to the higherkinetic activity commonly described by the Q10 rule where it isestimated that in the case of an enzyme the turnover doubles for everyincrease of 10 degrees Celsius.

In addition, there exist examples of natural mutations that destabilizeproteins at their normal operating conditions. Certain mutants can beactive at a lower temperature, but at a reduced level compared to thewild type proteins. This is also typically described by a reduction inactivity as guided by the Q10 or similar rules.

It is desirable to generate useful molecules that are conditionallyactivated. For example, it is desirable to generate molecules that arevirtually inactive at wild-type operating conditions but are active atother than wild-type operating conditions at a level that is equal to orbetter than at wild-type operating conditions, or that are activated orinactivated in certain microenvironments, or that are activated orinactivated over time. Besides temperature, other conditions for whichthe proteins can be evolved or optimized include pH, osmotic pressure,osmolality, oxidative stress and electrolyte concentration. Otherdesirable properties that can be optimized during evolution includechemical resistance, and proteolytic resistance.

Many strategies for evolving or engineering molecules have beenpublished. However, engineering or evolving a protein to be inactive orvirtually inactive (less than 10% activity and preferably less than 1%activity) at a wild type operating condition, while maintaining activityequivalent or better than its corresponding wild type protein at acondition other than a wild-type operating condition, requires thatdestabilizing mutation(s) co-exist with activity increasing mutationsthat do not counter the destabilizing effect. It is expected thatdestabilization would reduce the protein's activity greater than theeffects predicted by standard rules such as Q10. Therefore, the abilityto evolve proteins that work efficiently at lower temperature, forexample, while being inactivated under the normal operating conditionfor the corresponding wild-type protein, creates an unexpected new classof proteins.

Chimeric antigen receptors (CARs) have been used in treating cancers. US2013/0280220 discloses methods and compositions providing improved cellsencoding a chimeric antigen receptor that is specific for two or moreantigens, including tumor antigens. Cells expressing the chimericantigen receptor may be used in cell therapy. Such cell therapy may besuitable for any medical condition, although in specific embodiments thecell therapy is for cancer, including cancer involving solid tumors.

The present invention provides engineered conditionally active chimericantigen receptors that are inactive or less active at a normalphysiological condition but active at an aberrant physiologicalcondition.

Throughout this application, various publications are referenced byauthor and date. The disclosures of these publications are herebyincorporated by reference in their entireties into this application inorder to more fully describe the state of the art as known to thoseskilled therein as of the date of the disclosure described and claimedherein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a schematic representation of a chimeric antigen receptorin accordance with one embodiment of the present invention. ASTR is anantigen-specific targeting region, L is a linker, ESD is anextracellular spacer domain, TM is a transmembrane domain, CSD is aco-stimulatory domain, and ISD is an intracellular signaling domain.

FIGS. 2 and 3 show that expressing the conditionally active antibodiesof Example 1 as bivalent or monovalent antibodies does not significantlyalter that selectivity of these antibodies under pH 6.0 and over pH 7.4.

FIG. 4 is a profile of a size exclusive chromatograph indicating thatthe conditionally active antibodies of Example 2 do not aggregate.

FIG. 5 shows on and off rates for the conditionally active antibodies ofExample 2 as measured by a surface plasmon resonance (SPR) assay.

FIGS. 6A-6B show the selectivity of the conditionally active antibodiesas measured by the SPR assay of Example 2.

FIG. 7A shows that CAR-T cells had no effect on a population of CHOcells that do not express the target antigen X1 of the CAR-T cells. TheCAR molecule in the CAR-T cells of this example included an antibodyagainst target antigen X1, though this antibody was not conditionallyactive (Comparative Example A).

FIG. 7B shows that CAR-T cells reduced the population of CHO-63 cellsthat express the target antigen X1 of the CAR-T cells. These CAR-T cellsare the same cells as were used to generate the data shown in FIG. 7A(Comparative Example A).

FIG. 8A shows that CAR-T cells had no effect on a population of CHOcells that do not express the target antigen X1 of the CAR-T cells. TheCAR molecule in the CAR-T cells of this Example 3 included aconditionally active antibody against target antigen X1.

FIG. 8B shows that CAR-T cells reduced the population of CHO-63 cellsthat express the target antigen X1 of the CAR-T cells as tested inExample 3. These CAR-T cells are the same cells as were used to generatethe data shown in FIG. 8A.

FIGS. 9A-9B show cytokine release induced by binding of CAR-T cells withthe target antigen X1, as described in Example 3.

FIG. 10 shows conditionally active antibodies against target antigen X2.

FIG. 11A shows the cytotoxic effect induced by CAR-T cells binding toDaudi cells that express target antigen X2 and the cytotoxic effectinduced by CAR-T cells on HEK293 cells that do not express targetantigen X2, as described in Example 4.

FIGS. 12A-12B show cytokine release induced by binding of CAR-T cellswith the target antigen X1, as described in Example 5.

FIGS. 13A-13B show cytokine release induced by binding of CAR-T cellswith the target antigen X2, as described in Example 5.

FIG. 14 shows conditionally active antibodies against target antigen X3that are suitable for construction of CAR-T cells.

SUMMARY OF THE DISCLOSURE

In one aspect, the present invention provides a chimeric antigenreceptor (CAR) for binding with a target antigen. The CAR includes atleast one antigen specific targeting region. The CAR is an antibodyevolved from a wild-type antibody or a fragment thereof. The CARincludes a transmembrane domain; an intracellular signaling domain; andhas at least one of: (a) a decrease in binding activity to the targetantigen in an assay at a normal physiological condition compared to thewild-type antibody or a fragment thereof, and (b) an increase in bindingactivity to the target antigen in an assay at an aberrant conditioncompared to the wild-type antibody or a fragment thereof. The normal andaberrant conditions are a same condition having different values.

In yet another aspect, the present invention provides a chimeric antigenreceptor for binding with a target antigen. The CAR includes at leastone antigen specific targeting region evolved from a wild-type proteinor a domain thereof. The CAR also has at least one of: (a) a decrease inactivity in an assay at a normal physiological condition compared to theantigen specific targeting region of the wild-type protein or a domainthereof, and (b) an increase in activity in an assay under an aberrantcondition compared to the antigen specific targeting region of thewild-type protein or a domain thereof. The normal and aberrantconditions are a same condition having different values. The aberrantcondition is a condition present in a tumor microenvironment, a brainextracellular fluid, a stem cell niche, a lymph node, a tonsil, anadenoid, a sinus, or a synovial fluid. The CAR further comprises atransmembrane domain and an intracellular signaling domain.

In yet another aspect, the present invention provides a chimeric antigenreceptor for binding with a target antigen. The CAR includes atransmembrane domain; an intracellular signaling domain and at least twoantigen specific targeting regions connected by a conditional linker.The conditional linker has a first conformation at an aberrant conditionthat presents the at least two antigen specific targeting regions in amanner which has a higher binding affinity to the target antigen than abinding affinity of a second conformation of the same conditional linkerthat occurs at a normal physiological condition.

In yet another aspect, the chimeric antigen receptor has one or both oftwo antigen specific targeting regions evolved from a wild-type proteinor a domain thereof. The CAR also has at least one of: (a) a decrease inactivity in an assay at a normal physiological condition compared to theantigen specific targeting region of the wild-type protein or a domainthereof, and (b) an increase in activity in an assay under an aberrantcondition compared to the antigen specific targeting region of thewild-type protein or a domain thereof.

In yet another aspect, the present invention provides a chimeric antigenreceptor for binding with a target antigen The CAR includes atransmembrane domain; an intracellular signaling domain; and at leastone antigen specific targeting region that binds with the targetantigen; an extracellular spacer domain having a first conformation atan aberrant condition for the at least one antigen specific targetingregion that provides a higher binding affinity to the target antigenthan a second conformation of the antigen specific extracellular spacerdomain that occurs at a normal physiological condition.

In yet another aspect, the chimeric antigen receptor has an antigenspecific targeting region evolved from a wild-type protein or a domainthereof. The CAR also has at least one of: (a) a decrease in activity inan assay at a normal physiological condition compared to the antigenspecific targeting region of the wild-type protein or a domain thereof,and (b) an increase in activity in an assay under an aberrant conditioncompared to the antigen specific targeting region of the wild-typeprotein or a domain thereof. The normal and aberrant conditions are asame condition having different values.

In yet another aspect, the present invention provides a chimeric antigenreceptor for binding with a target antigen. The CAR includes at leastone antigen specific targeting region that binds with the targetantigen; an extracellular spacer domain having an enhancedubiquitylation-resistance at an aberrant condition than at a normalphysiological condition; a transmembrane domain; and an intracellularsignaling domain.

In another aspect, the present invention provides an expression vector,including a polynucleotide sequence encoding the chimeric antigenreceptor of the invention. The expression vector is selected fromlentivirus vectors, gamma retrovirus vectors, foamy virus vectors, adenoassociated virus vectors, adenovirus vectors, pox virus vectors, herpesvirus vectors, engineered hybrid viruses, and transposon mediatedvectors.

In yet another aspect, the present invention provides a geneticallyengineered cytotoxic cell that includes a polynucleotide sequenceencoding the chimeric antigen receptor of the invention. The cytotoxiccell may be a T cell and may be selected from a naive T cell, a centralmemory T cell, and an effector memory T cell.

In yet another aspect, the present invention provides a pharmaceuticalcomposition, including the chimeric antigen receptor, the expressionvector, and/or the genetically engineered cytotoxic cell of theinvention, and a pharmaceutically acceptable excipient.

In yet another aspect, the present invention provides a method fortreating a cancer in a subject, including the step of introducing anexpression vector including a polynucleotide sequence encoding thechimeric antigen receptor of the invention into a cytotoxic cellobtained from the subject to produce a genetically engineered cytotoxiccell; and administering the genetically engineered cytotoxic cell to thesubject.

Definitions

In order to facilitate understanding of the examples provided herein,certain frequently occurring methods and/or terms will be definedherein.

As used herein in connection with a measured quantity, the term “about”refers to the normal variation in that measured quantity that would beexpected by the skilled artisan making the measurement and exercising alevel of care commensurate with the objective of the measurement and theprecision of the measuring equipment used. Unless otherwise indicated,“about” refers to a variation of +/−10% of the value provided.

The term “agent” is used herein to denote a chemical compound, a mixtureof chemical compounds, an array of spatially localized compounds (e.g.,a VLSIPS peptide array, polynucleotide array, and/or combinatorial smallmolecule array), biological macromolecule, a bacteriophage peptidedisplay library, a bacteriophage antibody (e.g., scFv) display library,a polysome peptide display library, or an extract made from biologicalmaterials such as bacteria, plants, fungi, or animal (particularmammalian) cells or tissues. Agents are evaluated for potential enzymeactivity by inclusion in screening assays described herein below. Agentsare evaluated for potential activity as conditionally active biologictherapeutic enzymes by inclusion in screening assays described hereinbelow.

The term “amino acid” as used herein refers to any organic compound thatcontains an amino group (—NH₂) and a carboxyl group (—COOH); preferablyeither as free groups or alternatively after condensation as part ofpeptide bonds. The “twenty naturally encoded polypeptide-formingalpha-amino acids” are understood in the art and refer to: alanine (alaor A), arginine (arg or R), asparagine (asn or N), aspartic acid (asp orD), cysteine (cys or C), gluatamic acid (glu or E), glutamine (gin orQ), glycine (gly or G), histidine (his or H), isoleucine (ile or I),leucine (leu or L), lysine (lys or K), methionine (met or M),phenylalanine (phe or F), proline (pro or P), serine (ser or S),threonine (thr or T), tryptophan (tip or W), tyrosine (tyr or Y), andvaline (val or V).

The term “amplification” as used herein means that the number of copiesof a polynucleotide is increased.

The term “antibody” as used herein refers to intact immunoglobulinmolecules, as well as fragments of immunoglobulin molecules, such asFab, Fab′, (Fab′)2, Fv, and SCA fragments, that are capable of bindingto an epitope of an antigen. These antibody fragments, which retain someability to selectively bind to an antigen (e.g., a polypeptide antigen)of the antibody from which they are derived, can be made using wellknown methods in the art (see, e.g., Harlow and Lane, supra), and aredescribed further, as follows. Antibodies can be used to isolatepreparative quantities of the antigen by immunoaffinity chromatography.Various other uses of such antibodies are to diagnose and/or stagedisease (e.g., neoplasia) and for therapeutic application to treatdisease, such as for example: neoplasia, autoimmune disease, AIDS,cardiovascular disease, infections, and the like. Chimeric, human-like,humanized or fully human antibodies are particularly useful foradministration to human patients.

An Fab fragment consists of a monovalent antigen-binding fragment of anantibody molecule, and can be produced by digestion of a whole antibodymolecule with the enzyme papain, to yield a fragment consisting of anintact light chain and a portion of a heavy chain.

An Fab′ fragment of an antibody molecule can be obtained by treating awhole antibody molecule with pepsin, followed by reduction, to yield amolecule consisting of an intact light chain and a portion of a heavychain. Two Fab′ fragments are obtained per antibody molecule treated inthis manner.

An (Fab′)2 fragment of an antibody can be obtained by treating a wholeantibody molecule with the enzyme pepsin, without subsequent reduction.A (Fab′)2 fragment is a dimer of two Fab′ fragments, held together bytwo disulfide bonds.

An Fv fragment is defined as a genetically engineered fragmentcontaining the variable region of a light chain and the variable regionof a heavy chain expressed as two chains.

The term “antigen” or “Ag” as used herein is defined as a molecule thatprovokes an immune response. This immune response may involve eitherantibody production, or the activation of specificimmunologically-competent cells, or both. A person skilled in the artwill understand that any macromolecule, including virtually all proteinsor peptides, can serve as an antigen. Furthermore, antigens can bederived from recombinant or genomic DNA. A person skilled in the artwill understand that any DNA, which includes a nucleotide sequence or apartial nucleotide sequence encoding a protein that elicits an immuneresponse therefore encodes an “antigen” as that term is used herein.Furthermore, one skilled in the art will understand that an antigen neednot be encoded solely by a full length nucleotide sequence of a gene. Itis readily apparent that the present invention includes, but is notlimited to, the use of partial nucleotide sequences of more than onegene and that these nucleotide sequences are arranged in variouscombinations to elicit the desired immune response. Moreover, a skilledperson will understand that an antigen need not be encoded by a “gene”at all. It is readily apparent that an antigen can be generated,synthesized or can be derived from a biological sample. Such abiological sample can include, but is not limited to a tissue sample, atumor sample, a cell or a biological fluid.

“Antigen loss escape variants” as used herein refer to cells whichexhibit reduced or loss of expression of the target antigen, whichantigens are targeted by the CARs of the invention.

The term “autoimmune disease” as used herein is defined as a disorderthat results from an autoimmune response. An autoimmune disease is theresult of an inappropriate and excessive response to a self-antigen.Examples of autoimmune diseases include but are not limited to,Addison's disease, alopecia greata, ankylosing spondylitis, autoimmunehepatitis, autoimmune parotitis, Crohn's disease, diabetes (Type 1),dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis,Graves' disease, Guillain-Barr syndrome, Hashimoto's disease, hemolyticanemia, systemic lupus erythematosus, multiple sclerosis, myastheniagravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoidarthritis, sarcoidosis, scleroderma, Sjogren's syndrome,spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxedema,pernicious anemia, ulcerative colitis, among others.

The term “autologous,” as used herein refers to any material derivedfrom the same individual to which it is later to be reintroduced. Forexample, T cells from a patient may be isolated, genetically engineeredto express a CAR and then reintroduced into the patient.

The term “B-cell associated diseases” as used herein include B-cellimmunodeficiencies, autoimmune diseases and/or excessive/uncontrolledcell proliferation associated with B-cells (including lymphomas and/orleukemia's). Examples of such diseases, wherein bispecific CARs of theinvention may be used for therapeutic approaches include but are notlimited to systemic lupus erythematosus (SLE), diabetes, rheumatoidarthritis (RA), reactive arthritis, multiple sclerosis (MS), pemphigusvulgaris, celiac disease, Crohn's disease, inflammatory bowel disease,ulcerative colitis, autoimmune thyroid disease, X-linkedagammaglobulinaemis, pre-B acute lymphoblastic leukemia, systemic lupuserythematosus, common variable immunodeficiency, chronic lymphocyticleukemia, diseases associated with selective IgA deficiency and/or IgGsubclass deficiency, B lineage lymphomas (Hodgkin's lymphoma and/ornon-Hodgkin's lymphoma), immunodeficiency with thymoma, transienthypogammaglobulinemia and/or hyper IgM syndrome, as well asvirally-mediated B-cell diseases such as EBV mediatedlymphoproliferative disease, and chronic infections in which B-cellsparticipate in the pathophysiology.

The term “blood-brain barrier” or “BBB” refers to the physiologicalbarrier between the peripheral circulation and the brain and spinal cordwhich is formed by tight junctions within the brain capillaryendothelial plasma membranes, creating a tight barrier that restrictsthe transport of molecules into the brain, even very small moleculessuch as urea (60 Daltons). The blood-brain barrier within the brain, theblood-spinal cord barrier within the spinal cord, and the blood-retinalbarrier within the retina are contiguous capillary barriers within thecentral nerve system (CNS), and are herein collectively referred to asthe “blood-brain barrier” or “BBB.” The BBB also encompasses theblood-cerebral spinal fluid barrier (choroid plexus) where the barrieris included of ependymal cells rather than capillary endothelial cells.

The terms “cancer” and “cancerous” as used herein refer to or describethe physiological condition in mammals that is typically characterizedby unregulated cell growth. Examples of cancer include, but are notlimited to B-cell lymphomas (Hodgkin's lymphomas and/or non-Hodgkinslymphomas), brain tumor, breast cancer, colon cancer, lung cancer,hepatocellular cancer, gastric cancer, pancreatic cancer, cervicalcancer, ovarian cancer, liver cancer, bladder cancer, cancer of theurinary tract, thyroid cancer, renal cancer, carcinoma, melanoma, headand neck cancer, brain cancer, and prostate cancer, including but notlimited to androgen-dependent prostate cancer and androgen-independentprostate cancer.

The term “chimeric antigen receptor” or “CAR” or “CARs” as used hereinrefers to engineered receptors, which graft antigen specificity onto acytotoxic cell, for example T cells, NK cells and macrophages. The CARsof the invention may include at least one antigen specific targetingregion (ASTR), an extracellular spacer domain (ESD), a transmembranedomain (TM), one or more co-stimulatory domains (CSD), and anintracellular signaling domain (ISD). In an embodiment, the ESD and/orCSD are optional. In another embodiment, the CAR is a bispecific CAR,which is specific to two different antigens or epitopes. After the ASTRbinds specifically to a target antigen, the ISD activates intracellularsignaling. For example, the ISD can redirect T cell specificity andreactivity toward a selected target in a non-MHC-restricted manner,exploiting the antigen-binding properties of antibodies. Thenon-MHC-restricted antigen recognition gives T cells expressing the CARthe ability to recognize an antigen independent of antigen processing,thus bypassing a major mechanism of tumor escape. Moreover, whenexpressed in T cells, CARs advantageously do not dimerize withendogenous T cell receptor (TCR) alpha and beta chains.

The term “co-express” as used herein refers to simultaneous expressionof two or more genes. Genes may be nucleic acids encoding, for example,a single protein or a chimeric protein as a single polypeptide chain.For example, the CARs of the invention may be co-expressed with atherapeutic control (for example truncated epidermal growth factor(EGFRt)), wherein the CAR is encoded by a first polynucleotide chain andthe therapeutic control is encoded by a second polynucleotide chain. Inan embodiment, the first and second polynucleotide chains are linked bya nucleic acid sequence that encodes a cleavable linker. Alternately,the CAR and the therapeutic control are encoded by two differentpolynucleotides that are not linked via a linker but are instead encodedby, for example, two different vectors.

The term “cognate” as used herein refers to a gene sequence that isevolutionarily and functionally related between species. For example,but without limitation, in the human genome the human CD4 gene is thecognate gene to the mouse 3d4 gene, since the sequences and structuresof these two genes indicate that they are highly homologous and bothgenes encode a protein which functions in signaling T cell activationthrough MHC class II-restricted antigen recognition.

The term “conditionally active biologic protein” refers to a variant, ormutant, of a wild-type protein which is more or less active than theparent wild-type protein under one or more normal physiologicalconditions. This conditionally active protein also exhibits activity inselected regions of the body and/or exhibits increased or decreasedactivity under aberrant, or permissive, physiological conditions. Theterm “normal physiological condition” as used herein refers to one oftemperature, pH, osmotic pressure, osmolality, oxidative stress,electrolyte concentration, a concentration of a small organic moleculesuch as glucose, lactic acid, pyruvate, nutrient components, othermetabolites, and the like, a concentration of another molecule such asoxygen, carbonate, phosphate, and carbon dioxide, as well as cell types,and nutrient availability, which would be considered within a normalrange at the site of administration, or at the tissue or organ at thesite of action, to a subject.

The term “aberrant condition” as used herein refers to a condition thatdeviates from the normally acceptable range for that condition. In oneaspect, the conditionally active biologic protein is virtually inactiveat a normal physiological condition but is active at an aberrantcondition at a level that is equal or better than the wild-type proteinfrom which it is derived. For example, in one aspect, an evolvedconditionally active biologic protein is virtually inactive at bodytemperature, but is active at lower temperatures. In another aspect, theconditionally active biologic protein is reversibly or irreversiblyinactivated at the normal physiological condition. In a further aspect,the wild-type protein is a therapeutic protein. In another aspect, theconditionally active biologic protein is used as a drug, or therapeuticagent. In yet another aspect, the protein is more or less active inhighly oxygenated blood, such as, for example, after passage through thelung or in the lower pH environments found in the kidney.

“Conservative amino acid substitutions” refer to the interchangeabilityof residues having similar side chains. For example, a group of aminoacids having aliphatic side chains is glycine, alanine, valine, leucine,and isoleucine; a group of amino acids having aliphatic-hydroxyl sidechains is serine and threonine; a group of amino acids havingamide-containing side chains is asparagine and glutamine; a group ofamino acids having aromatic side chains is phenylalanine, tyrosine, andtryptophan; a group of amino acids having basic side chains is lysine,arginine, and histidine; and a group of amino acids havingsulfur-containing side chains is cysteine and methionine. Preferredconservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, and asparagine-glutamine.

The term “corresponds to” is used herein to mean that a polynucleotidesequence is homologous (i.e., is identical, not strictly evolutionarilyrelated) to all or a portion of a reference polynucleotide sequence, orthat a polypeptide sequence is identical to a reference polypeptidesequence. In contradistinction, the term “complementary to” is usedherein to mean that the complementary sequence is homologous to all or aportion of a reference polynucleotide sequence. For illustration, thenucleotide sequence “TATAC” corresponds to a reference “TATAC” and iscomplementary to a reference sequence “GTATA.”

The term “co-stimulatory ligand” as used herein includes a molecule onan antigen presenting cell (e.g., dendritic cell, B cell, and the like)that specifically binds a cognate co-stimulatory molecule on a T cell,thereby providing a signal which, in addition to the primary signalprovided by, for instance, by the binding of a TCR/CD3 complex with anMHC molecule loaded with peptide, mediates a T cell response, including,but not limited to, proliferation, activation, differentiation, and thelike. A co-stimulatory ligand can include, but is not limited to, CD7,B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, an induciblecostimulatory ligand (ICOS-L), an intercellular adhesion molecule(ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, a lymphotoxinbeta receptor, 3/TR6, ILT3, ILT4, HVEM, an agonist or an antibody thatbinds to a Toll ligand receptor and a ligand that specifically bindswith B7-H3. A co-stimulatory ligand also encompasses, inter alia, anantibody that specifically binds with a co-stimulatory molecule presenton a T cell, such as, but not limited to, CD27, CD28, 4-1BB, OX40, CD30,CD40, PD-1, ICOS, a lymphocyte function-associated antigen-1 (LFA-1),CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds withCD83.

The term “co-stimulatory molecule” as used herein refers to the cognatebinding partner on a T cell that specifically binds with aco-stimulatory ligand, thereby mediating a co-stimulatory response bythe T cell, such as, but not limited to, proliferation. Co-stimulatorymolecules include, but are not limited to an MHC class 1 molecule, BTLAand a Toll ligand receptor.

The term “co-stimulatory signal” as used herein refers to a signal,which in combination with a primary signal, such as TCR/CD3 ligation,leads to T cell proliferation and/or upregulation or down regulation ofkey molecules.

The term “cytotoxic cell” as used herein means a cell which can injureor destroy invading microorganisms, tumor cells or other diseased tissuecells. This term is meant to include natural killer (NK) cells,activated NK cells, neutrophils, T cells, eosinophils, basophils,B-cells, macrophages and lymphokine-activated killer (LAK) cells amongother cell types. The cytotoxic cell, through an antibody, receptor,ligand or fragments/derivatives thereof, is bound to a target cell toform a stable complex, and stimulates the cytotoxic cell to destroy thetarget cell.

Cytotoxic cells may also include other immune cells with tumor lyticcapabilities including but not limited to natural killer T cells (Heczeyet al., “Invariant NKT cells with chimeric antigen receptor provide anovel platform for safe and effective cancer immunotherapy,” Blood, vol.124, pp. 2824-2833, 2014) and granulocytes. Further, cytotoxic cells mayinclude immune cells with phagocytic capability including but notlimited to macrophages and granulocytes, cells with stem cell and/orprogenitor cell properties including, but not limited to, hematopoieticstem/progenitor cells (Zhen et al., “HIV-specific Immunity Derived FromChimeric Antigen Receptor-engineered Stem Cells,” Mol Ther., vol. 23,pp. 1358-1367, 2015), embryonic stem cells (ESCs), cord blood stemcells, and induced pluripotent stem cells (iPSCs) (Themeli et al., “Newcell sources for T cell engineering and adoptive immunotherapy,” CellStem Cell., vol. 16, pp. 357-366, 2015). Additionally, cytotoxic cellsinclude “synthetic cells” such as iPSC-derived T cells (TiPSCs) (Themeliet al., “Generation of tumor-targeted human T lymphocytes from inducedpluripotent stem cells for cancer therapy,” Nat Biotechnol., vol. 31,pp. 928-933, 2013) or iPSC-derived NK cells.

The term “degrading effective” amount refers to the amount of enzymewhich is required to process at least 50% of the substrate, as comparedto substrate not contacted with the enzyme.

The term “directional ligation” refers to a ligation in which a 5′ endand a 3′ end of a polynucleotide are different enough to specify apreferred ligation orientation. For example, an otherwise untreated andundigested PCR product that has two blunt ends will typically not have apreferred ligation orientation when ligated into a cloning vectordigested to produce blunt ends in its multiple cloning site; thus,directional ligation will typically not be displayed under thesecircumstances. In contrast, directional ligation will typically bedisplayed when a digested PCR product having a 5′ EcoR I-treated end anda 3′ BamH I is ligated into a cloning vector that has a multiple cloningsite digested with EcoR I and BamH I.

The term “disease targeted by genetically modified cytotoxic cells” asused herein encompasses the targeting of any cell involved in any mannerin any disease by the genetically modified cells of the invention,irrespective of whether the genetically modified cells target diseasedcells or healthy cells to effectuate a therapeutically beneficialresult. The genetically modified cells include but are not limited togenetically modified T cells, NK cells, and macrophages. The geneticallymodified cells express the CARs of the invention, which CARs may targetany of the antigens expressed on the surface of target cells. Examplesof antigens which may be targeted include but are not limited toantigens expressed on B-cells; antigens expressed on carcinomas,sarcomas, lymphomas, leukemia, germ cell tumors, and blastomas; antigensexpressed on various immune cells; and antigens expressed on cellsassociated with various hematologic diseases, autoimmune diseases,and/or inflammatory diseases. Other antigens that may be targeted willbe apparent to those of skill in the art and may be targeted by the CARsof the invention in connection with alternate embodiments thereof.

The terms “genetically modified cells”, “redirected cells”, “geneticallyengineered cells” or “modified cells” as used herein refer to cells thatexpress the CARs of the invention.

The term “DNA shuffling” is used herein to indicate recombinationbetween substantially homologous but non-identical sequences, in someembodiments DNA shuffling may involve crossover via non-homologousrecombination, such as via cer/lox and/or flp/frt systems and the like.DNA shuffling can be random or non-random.

The term “drug” or “drug molecule” refers to a therapeutic agentincluding a substance having a beneficial effect on a human or animalbody when it is administered to the human or animal body. Preferably,the therapeutic agent includes a substance that can treat, cure orrelieve one or more symptoms, illnesses, or abnormal conditions in ahuman or animal body or enhance the wellness of a human or animal body.

An “effective amount” is an amount of a conditionally active biologicprotein or fragment which is effective to treat or prevent a conditionin a living organism to whom it is administered over some period oftime, e.g., provides a therapeutic effect during a desired dosinginterval.

The term “electrolyte” as used herein defines a mineral in the blood orother body fluids that carries a charge. For example, in one aspect, thenormal physiological condition and aberrant condition can be conditionsof “electrolyte concentration”. In one aspect, the electrolyteconcentration to be tested is selected from one or more of ionizedcalcium, sodium, potassium, magnesium, chloride, bicarbonate, andphosphate concentration. For example, in one aspect, normal range ofserum calcium is 8.5 to 10.2 mg/dL. In this aspect, aberrant serumcalcium concentration may be selected from either above or below thenormal range, m another example, in one aspect, normal range of serumchloride is 96-106 milliequivalents per liter (mEq/L). In this aspect,aberrant serum chloride concentration may be selected from either aboveor below the normal range, in another example, in one aspect, a normalrange of serum magnesium is from 1.7-2.2 mg/dL. In this aspect, anaberrant serum magnesium concentration may be selected from either aboveor below the normal range, in another example, in one aspect, a normalrange of serum phosphorus is from 2.4 to 4.1 mg/dL. In this aspect,aberrant serum phosphorus concentration may be selected from eitherabove or below the normal range. In another example, in one aspect, anormal range of serum, or blood, sodium is from 135 to 145 mEq/L. Inthis aspect, aberrant serum, or blood, sodium concentration may beselected from either above or below the normal range. In anotherexample, in one aspect, a normal range of serum, or blood, potassium isfrom 3.7 to 5.2 mEq/L. In this aspect, aberrant serum, or blood,potassium concentration maybe selected from either above or below thenormal range. In a further aspect, a normal range of serum bicarbonateis from 20 to 29 mEq/L. In this aspect, aberrant serum, or blood,bicarbonate concentration may be selected from either above or below thenormal range. In a different aspect, bicarbonate levels can be used toindicate normal levels of acidity (pH), in the blood. The term“electrolyte concentration” may also be used to define the condition ofa particular electrolyte in a tissue or body fluid other than blood orplasma. In this case, the normal physiological condition is consideredto be the clinically normal range for that tissue or fluid. In thisaspect, aberrant tissue or fluid electrolyte concentration may beselected from either above or below the normal range.

The term “epitope” as used herein refers to an antigenic determinant onan antigen, such as an enzyme polypeptide, to which the paratope of anantibody, such as an enzyme-specific antibody, binds. Antigenicdeterminants usually consist of chemically active surface groupings ofmolecules, such as amino acids or sugar side chains, and can havespecific three-dimensional structural characteristics, as well asspecific charge characteristics. As used herein “epitope” refers to thatportion of an antigen or other macromolecule capable of forming abinding interaction that interacts with the variable region binding bodyof an antibody. Typically, such binding interaction is manifested as anintermolecular contact with one or more amino acid residues of a CDR.

As used herein, the term “evolution”, or “evolving”, refers to using oneor more methods of mutagenesis to generate a novel polynucleotideencoding a novel polypeptide, which novel polypeptide is itself animproved biological molecule &/or contributes to the generation ofanother improved biological molecule. In a particular non-limitingaspect, the present disclosure relates to evolution of conditionallyactive biologic proteins from a parent wild type protein. In one aspect,for example, evolution relates to a method of performing bothnon-stochastic polynucleotide chimerization and non-stochasticsite-directed point mutagenesis disclosed in U.S. patent applicationpublication 2009/0130718. More particularly, the present disclosureprovides methods for evolution of conditionally active biologic enzymeswhich exhibit reduced activity at normal physiological conditionscompared to a wild-type enzyme parent molecule, but enhanced activityunder one or more aberrant conditions compared to the antigen specifictargeting region of the wild-type enzyme.

The terms “fragment”, “derivative” and “analog” when referring to areference polypeptide include a polypeptide which retains at least onebiological function or activity that is at least essentially same asthat of the reference polypeptide. Furthermore, the terms “fragment”,“derivative” or “analog” are exemplified by a “pro-form” molecule, suchas a low activity proprotein that can be modified by cleavage to producea mature enzyme with significantly higher activity.

The term “gene” as used herein means the segment of DNA involved inproducing a polypeptide chain; it includes regions preceding andfollowing the coding region (leader and trailer) as well as interveningsequences (nitrons) between individual coding segments (exons).

The term “heterologous” as used herein means that one single-strandednucleic acid sequence is unable to hybridize to another single-strandednucleic acid sequence or its complement. Thus, areas of heterology meanthat areas of polynucleotides or polynucleotides have areas or regionswithin their sequence which are unable to hybridize to another nucleicacid or polynucleotide. Such regions or areas are for example areas ofmutations.

The term “homologous” or “homeologous” as used herein means that onesingle-stranded nucleic acid sequence may hybridize to a complementarysingle-stranded nucleic acid sequence. The degree of hybridization maydepend on a number of factors including the amount of identity betweenthe sequences and the hybridization conditions such as temperature andsalt concentrations as discussed later. Preferably the region ofidentity is greater than about 5 bp, more preferably the region ofidentity is greater than 10 bp.

The benefits of this disclosure extend to “industrial applications” (orindustrial processes), which term is used to include applications incommercial industry proper (or simply industry) as well asnon-commercial industrial applications (e.g. biomedical research at anon-profit institution). Relevant applications include those in areas ofdiagnosis, medicine, agriculture, manufacturing, and academia.

The term “immune cell” as used herein refers to cells of the mammalianimmune system including but not limited to antigen presenting cells,B-cells, basophils, cytotoxic T cells, dendritic cells, eosinophils,granulocytes, helper T cells, leukocytes, lymphocytes, macrophages, mastcells, memory cells, monocytes, natural killer cells, neutrophils,phagocytes, plasma cells and T cells.

The term “immune response” as used herein refers to immunities includingbut not limited to innate immunity, humoral immunity, cellular immunity,immunity, inflammatory response, acquired (adaptive) immunity,autoimmunity and/or overactive immunity

The term “isolated” as used herein means that the material is removedfrom its original environment (e.g., the natural environment if it isnaturally occurring). For example, a naturally-occurring polynucleotideor enzyme present in a living animal is not isolated, but the samepolynucleotide or enzyme, separated from some or all of the coexistingmaterials in the natural system, is isolated. Such polynucleotides couldbe part of a vector and/or such polynucleotides or enzymes could be partof a composition, and still be isolated in that such vector orcomposition is not part of its natural environment.

The term “isolated nucleic acid” as used herein to define a nucleicacid, e.g., a DNA or RNA molecule, that is not immediately contiguouswith the 5′ and 3′ flanking sequences with which it normally isimmediately contiguous when present in the naturally occurring genome ofthe organism from which it is derived. The term thus describes, forexample, a nucleic acid that is incorporated into a vector, such as aplasmid or viral vector; a nucleic acid that is incorporated into thegenome of a heterologous cell (or the genome of a homologous cell, butat a site different from that at which it naturally occurs); and anucleic acid that exists as a separate molecule, e.g., a DNA fragmentproduced by PCR amplification or restriction enzyme digestion, or an RNAmolecule produced by in vitro transcription. The term also describes arecombinant nucleic acid that forms part of a hybrid gene encodingadditional polypeptide sequences that can be used, for example, in theproduction of a fusion protein.

The term “lentivirus” as used herein refers to a genus of theRetroviridae family Lentiviruses are unique among the retroviruses inbeing able to infect non-dividing cells; they can deliver a significantamount of genetic information into the DNA of the host cell, so they areone of the most efficient ways to deliver a gene delivery vector. HIV,SIV, and FIV are all examples of lentiviruses. Vectors derived fromlentiviruses offer the means to achieve significant levels of genetransfer in vivo.

The term “ligand” as used herein refers to a molecule, such as a randompeptide or variable segment sequence that is recognized by a particularreceptor. As a person skilled in the art will recognize, a molecule (ormacromolecular complex) can be both a receptor and a ligand. In general,the binding partner having a smaller molecular weight is referred to asthe ligand and the binding partner having a greater molecular weight isreferred to as a receptor.

The term “ligation” as used herein refers to the process of formingphosphodiester bonds between two double stranded nucleic acid fragments(Sambrook et al., (1982). Molecular Cloning: A Laboratory Manual. ColdSpring Harbour Laboratory, Cold Spring Harbor, N.Y., p. 146; Sambrook etal., Molecular Cloning: a laboratory manual, 2^(nd) Ed., Cold SpringHarbor Laboratory Press, 1989). Unless otherwise provided, ligation maybe accomplished using known buffers and conditions with 10 units of T4DNA ligase (“ligase”) per 0.5 micrograms of approximately equimolaramounts of the DNA fragments to be ligated.

The terms “linker” or “spacer” as used herein refer to a molecule orgroup of molecules that connects two molecules, such as a DNA bindingprotein and a random peptide, and serves to place the two molecules in apreferred configuration, e.g., so that the random peptide can bind to areceptor with minimal steric hindrance from the DNA binding protein.“Linker” (L) or “linker domain” or “linker region” as used herein refersto an oligo- or polypeptide region of from about 1 to 100 amino acids inlength, which links together any of the domains/regions of the CARs ofthe invention. Linkers may be composed of flexible residues like glycineand serine so that the adjacent protein domains are free to moverelative to one another. Longer linkers may be used when it is desirableto ensure that two adjacent domains do not sterically interfere with oneanother. Linkers may be cleavable or non-cleavable. Examples ofcleavable linkers include 2A linkers (for example T2A), 2A-like linkersor functional equivalents thereof and combinations thereof. In someembodiments, the linkers include the picornaviral 2A-like linker, CHYSELsequences of porcine teschovirus (P2A), Thosea asigna virus (T2A) orcombinations, variants and functional equivalents thereof. Other linkerswill be apparent to those skilled in the art and may be used inconnection with alternate embodiments of the invention.

The term “mammalian cell surface display” as used herein refers to atechnique whereby a protein or antibody, or a portion of an antibody, isexpressed and displayed on a mammalian host cell surface for screeningpurposes; for example, by screening for specific antigen binding by acombination of magnetic beads and fluorescence-activated cell sorting.In one aspect, mammalian expression vectors are used for simultaneousexpression of immunoglobulins as both a secreted and cell surface boundform as in DuBridge et al., US 2009/0136950. In another aspect, thetechniques are employed for screening a viral vector encoding for alibrary of antibodies or antibody fragments that are displayed on thecell membranes when expressed in a cell as in Gao et al., US2007/0111260. Whole IgG surface display on mammalian cells is known. Forexample, Akamatsuu et al. developed a mammalian cell surface displayvector, suitable for directly isolating IgG molecules based on theirantigen-binding affinity and biological activity. Using an Epstein-Barrvirus-derived episomal vector, antibody libraries were displayed aswhole IgG molecules on the cell surface and screened for specificantigen binding by a combination of magnetic beads andfluorescence-activated cell sorting. Plasmids encoding antibodies withdesired binding characteristics were recovered from sorted cells andconverted to a form suitable for production of soluble IgG. SeeAkamatsuu et al. J. Immunol. Methods, vol. 327, pages 40-52, 2007. Ho etal. used human embryonic kidney 293T cells that are widely used fortransient protein expression for cell surface display of single-chain Fvantibodies for affinity maturation. Cells expressing a rare mutantantibody with higher affinity were enriched 240-fold by a single-passcell sorting from a large excess of cells expressing WT antibody with aslightly lower affinity. Furthermore, a highly enriched mutant wasobtained with increased binding affinity for CD22 after a singleselection of a combinatory library randomizing an intrinsic antibodyhotspot. See Ho et al., “Isolation of anti-CD22 Fv with high affinity byFv display on human cells,” Proc Natl Acad Sci USA, vol. 103, pages9637-9642, 2006.

B cells specific for an antigen may also be used. Such B cells may bedirectly isolated from peripheral blood mononuclear cells (PBMC) ofhuman donors. Recombinant, antigen-specific single-chain Fv (scFv)libraries are generated from this pool of B cells and screened bymammalian cell surface display by using a Sindbis virus expressionsystem. The variable regions (VRs) of the heavy chains (HCs) and lightchains (LCs) can be isolated from positive clones and recombinant fullyhuman antibodies produced as whole IgG or Fab fragments. In this manner,several hypermutated high-affinity antibodies binding the Q13 virus likeparticle (VLP), a model viral antigen, as well as antibodies specificfor nicotine can be isolated. See Beerli et al., “Isolation of humanmonoclonal antibodies by mammalian cell display,” Proc Natl Acad SciUSA, vol. 105, pages 14336-14341, 2008.

Yeast cell surface display may also be used in the present invention,for example, see Kondo and Ueda, “Yeast cell-surfacedisplay-applications of molecular display,” Appl. Microbiol.Biotechnol., vol. 64, pages 28-40, 2004, which describes for example, acell-surface engineering system using the yeast Saccharomycescerevisiae. Several representative display systems for the expression inyeast S. cerevisiae are described in Lee et al, “Microbial cell-surfacedisplay,” TRENDS in Bitechnol., vol. 21, pages 45-52, 2003. Also Boderand Wittrup, “Yeast surface display for screening combinatorialpolypeptide libraries,” Nature Biotechnol., vol. 15, pages 553, 1997.

The term “manufacturing” as used herein refers to production of aprotein in a sufficient quantity to permit at least Phase I clinicaltesting of a therapeutic protein, or sufficient quantity for regulatoryapproval of a diagnostic protein.

As used herein, the term “microenvironment” means any portion or regionof a tissue or body that has a constant or temporal, physical orchemical difference from other regions of the tissue or other regions ofthe body.

As used herein, the term “molecular property to be evolved” includesreference to molecules included of a polynucleotide sequence, moleculesincluded of a polypeptide sequence, and molecules included in part of apolynucleotide sequence and in part of a polypeptide sequence.Particularly relevant—but by no means limiting—examples of molecularproperties to be evolved include protein activities at specifiedconditions, such as related to temperature; salinity; osmotic pressure;pH; oxidative stress, and concentration of glycerol, DMSO, detergent,and/or any other molecular species with which contact is made in areaction environment. Additional particularly relevant—but by no meanslimiting—examples of molecular properties to be evolved includestabilities—e.g. the amount of a residual molecular property that ispresent after a specified exposure time to a specified environment, suchas may be encountered during storage.

The term “mutations” as used herein means changes in the sequence of awild-type nucleic acid sequence or changes in the sequence of a peptide.Such mutations may be point mutations such as transitions ortransversions. The mutations may be deletions, insertions orduplications.

The term “multispecific antibody” as used herein is an antibody havingbinding affinities for at least two different epitopes. Multispecificantibodies can be prepared as full-length antibodies or antibodyfragments (e.g. F(ab′)₂ bispecific antibodies). Engineered antibodiesmay bind to two, three or more (e.g. four) antigens (see, e.g., US2002/0004587 A1). One conditionally active antibody may be engineered tobe multispecific, or two antibodies may be engineered to include ahetero-dimer that binds to two antigens. Multispecific antibodies canalso be multifunctional.

As used herein, the degenerate “N,N,G/T” nucleotide sequence represents32 possible triplets, where “N” can be A, C, G or T.

The term “naturally-occurring” as used herein as applied to the objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory isnaturally occurring. Generally, the term naturally occurring refers toan object as present in a non-pathological (un-diseased) individual,such as would be typical for the species.

As used herein, “normal physiological conditions”, or “wild typeoperating conditions”, are those conditions of temperature, pH, osmoticpressure, osmolality, oxidative stress and electrolyte concentrationwhich would be considered within a normal range at the site ofadministration, or the site of action, in a subject.

As used herein, the term “nucleic acid molecule” is included of at leastone base or one base pair, depending on whether it is single-stranded ordouble-stranded, respectively. Furthermore, a nucleic acid molecule maybelong exclusively or chimerically to any group of nucleotide-containingmolecules, as exemplified by, but not limited to, the following groupsof nucleic acid molecules: RNA, DNA, genomic nucleic acids, non-genomicnucleic acids, naturally occurring and not naturally occurring nucleicacids, and synthetic nucleic acids. This includes, by way ofnon-limiting example, nucleic acids associated with any organelle, suchas the mitochondria, ribosomal RNA, and nucleic acid molecules includedchimerically of one or more components that are not naturally occurringalong with naturally occurring components.

Additionally, a “nucleic acid molecule” may contain in part one or morenon-nucleotide-based components as exemplified by, but not limited to,amino acids and sugars. Thus, by way of example, but not limitation, aribozyme that is in part nucleotide-based and in part protein-based isconsidered a “nucleic acid molecule”.

The terms “nucleic acid sequence coding for” or a “DNA coding sequenceof or a “nucleotide sequence encoding” as used herein refer to a DNAsequence which is transcribed and translated into an enzyme when placedunder the control of appropriate regulatory sequences such as promoters.A “promotor” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. The promoter is part of the DNA sequence.This sequence region has a start codon at its 3′ terminus. The promotersequence does include the minimum number of bases where elementsnecessary to initiate transcription at levels detectable abovebackground. However, after the RNA polymerase binds the sequence andtranscription is initiated at the start codon (3′ terminus with apromoter), transcription proceeds downstream in the 3′ direction. Withinthe promotor sequence will be found a transcription initiation site(conveniently defined by mapping with nuclease S1) as well as proteinbinding domains (consensus sequences) responsible for the binding of RNApolymerase.

The term “oligonucleotide” (or synonymously an “oligo”) refers to eithera single stranded polydeoxynucleotide or two complementarypolydeoxynucleotide strands which may be chemically synthesized. Suchsynthetic oligonucleotides may or may not have a 5′ phosphate. Thosethat do not will not ligate to another oligonucleotide without adding aphosphate with an ATP in the presence of a kinase. A syntheticoligonucleotide will ligate to a fragment that has not beendephosphorylated.

As used herein, the term “operably linked” refers to a linkage ofpolynucleotide elements in a functional relationship. A nucleic acid is“operably linked” when it is placed into a functional relationship withanother nucleic acid sequence. For instance, a promoter or enhancer isoperably linked to a coding sequence if it affects the transcription ofthe coding sequence. Operably linked means that the DNA sequences beinglinked are typically contiguous and, where necessary to join two proteincoding regions, contiguous and in reading frame.

A coding sequence is “operably linked to” another coding sequence whenRNA polymerase will transcribe the two coding sequences into a singlemRNA, which is then translated into a single polypeptide having aminoacids derived from both coding sequences. The coding sequences need notbe contiguous to one another so long as the expressed sequences areultimately processed to produce the desired protein.

As used herein the term “parental polynucleotide set” is a set includedof one or more distinct polynucleotide species. Usually this term isused in reference to a progeny polynucleotide set which is preferablyobtained by mutagenization of the parental set, in which case the terms“parental”, “starting” and “template” are used interchangeably.

The term “patient”, or “subject”, refers to an animal, for example amammal, such as a human, who is the object of treatment. The subject, orpatient, may be either male or female.

As used herein the term “physiological conditions” refers totemperature, pH, osmotic pressure, ionic strength, viscosity, and likebiochemical parameters which are compatible with a viable organism,and/or which typically exist intracellularly in a viable cultured yeastcell or mammalian cell. For example, the intracellular conditions in ayeast cell grown under typical laboratory culture conditions arephysiological conditions. Suitable in vitro reaction conditions for invitro transcription cocktails are generally physiological conditions. Ingeneral, in vitro physiological conditions include 50-200 mM NaCl orKCl, pH 6.5-8.5, 20-45 degrees C. and 0.001-10 mM divalent cation (e.g.,Mg^(++″), Ca⁺⁺); preferably about 150 mM NaCl or KCl, pH 7.2-7.6, 5 mMdivalent cation, and often include 0.01-1.0 percent nonspecific protein(e.g., bovine serum albumin (BSA)). A non-ionic detergent (Tween, NP-40,Triton X-100) can often be present, usually at about 0.001 to 2%,typically 0.05-0.2% (v/v). Particular aqueous conditions may be selectedby the practitioner according to conventional methods. For generalguidance, the following buffered aqueous conditions may be applicable:10-250 mM NaCl, 5-50 mM Tris HCl, pH 5-8, with optional addition ofdivalent cation(s) and/or metal chelators and/or non-ionic detergentsand/or membrane fractions and/or anti-foam agents and/or scintillants.Normal physiological conditions refer to conditions of temperature, pH,osmotic pressure, osmolality, oxidative stress and electrolyteconcentration in vivo in a patient or subject at the site ofadministration, or the site of action, which would be considered withinthe normal range in a patient.

Standard convention (5′ to 3′) is used herein to describe the sequenceof double stranded polynucleotides.

The term “population” as used herein means a collection of componentssuch as polynucleotides, portions or polynucleotides or proteins. A“mixed population” means a collection of components which belong to thesame family of nucleic acids or proteins (i.e., are related) but whichdiffer in their sequence (i.e., are not identical) and hence in theirbiological activity.

A molecule having a “pro-form” refers to a molecule that undergoes anycombination of one or more covalent and noncovalent chemicalmodifications (e.g. glycosylation, proteolytic cleavage, dimerization oroligomerization, temperature-induced or pH-induced conformationalchange, association with a co-factor, etc.) en route to attain a moremature molecular form having a property difference (e.g. an increase inactivity) in comparison with the reference pro-form molecule. When twoor more chemical modifications (e.g. two proteolytic cleavages, or aproteolytic cleavage and a deglycosylation) can be distinguished enroute to the production of a mature molecule, the reference precursormolecule may be termed a “pre-pro-form” molecule.

As used herein, the term “receptor” refers to a molecule that has anaffinity for a given ligand. Receptors can be naturally occurring orsynthetic molecules. Receptors can be employed in an unaltered state oras aggregates with other species. Receptors can be attached, covalentlyor non-covalently, to a binding member, either directly or via aspecific binding substance. Examples of receptors include, but are notlimited to, antibodies, including monoclonal antibodies and antiserareactive with specific antigenic determinants (such as on viruses,cells, or other materials), cell membrane receptors, complexcarbohydrates and glycoproteins, enzymes, and hormone receptors.

The term “reductive reassortment”, as used herein, refers to theincrease in molecular diversity that is accrued through deletion (and/orinsertion) events that are mediated by repeated sequences.

The term “restriction site” as used herein refers to a recognitionsequence that is necessary for the manifestation of the action of arestriction enzyme, and includes a site of catalytic cleavage. It isappreciated that a site of cleavage may or may not be contained within aportion of a restriction site that includes a low ambiguity sequence(i.e. a sequence containing the principal determinant of the frequencyof occurrence of the restriction site). When an enzyme (e.g. arestriction enzyme) is said to “cleave” a polynucleotide, it isunderstood to mean that the restriction enzyme catalyzes or facilitatesa cleavage of a polynucleotide.

As used herein, the term “single-chain antibody” refers to a polypeptideincluding a VH domain and a VL domain in polypeptide linkage, generallyliked via a spacer peptide, and which may include additional amino acidsequences at the amino- and/or carboxy-termini. For example, asingle-chain antibody may include a tether segment for linking to theencoding polynucleotide. As an example a scFv is a single-chainantibody. Single-chain antibodies are generally proteins consisting ofone or more polypeptide segments of at least 10 contiguous aminosubstantially encoded by genes of the immunoglobulin superfamily (e.g,see The Immunoglobulin Gene Superfamily, A. F. Williams and A. N.Barclay, in Immunoglobulin Genes, T. Honjo, F. W. Alt, and THE. Rabbits,eds., (1989) Academic press: San Diego, Calif., pp. 361-368, mostfrequently encoded by a rodent, non-human primate, avian, porcinebovine, ovine, goat, or human heavy chain or light chain gene sequence.A functional single-chain antibody generally contains a sufficientportion of an immunoglobulin superfamily gene product so as to retainthe property of binding to a specific target molecule, typically areceptor or antigen (epitope).

The members of a pair of molecules (e.g., an antibody-antigen pair andligand-receptor pair) are said to “specifically bind” to each other ifthey bind to each other with greater affinity than to other,non-specific molecules. For example, an antibody raised against anantigen to which it binds more efficiently than to a non-specificprotein can be described as specifically binding to the antigen.

The term “stimulation” as used herein means a primary response inducedby binding of a stimulatory molecule (e.g., a TCR/CD3 complex) with itscognate ligand thereby mediating a signal transduction event, such as,but not limited to, signal transduction via the TCR/CD3 complex.Stimulation can mediate altered expression of certain molecules, such asdownregulation of TGF-β, and/or reorganization of cytoskeletalstructures, and the like.

The term “stimulatory molecule” as used herein means a molecule on a Tcell that specifically binds with a cognate stimulatory ligand presenton an antigen presenting cell.

The term “stimulatory ligand” as used herein means a ligand that whenpresent on an antigen presenting cell (e.g, a dendritic cell, a B-cell,and the like) can specifically bind with a cognate binding partner(referred to herein as a “stimulatory molecule”) on a T cell, therebymediating a primary response by the T cell, including, but not limitedto, activation, initiation of an immune response, proliferation, and thelike. Stimulatory ligands are well-known in the art and encompass, interalia, an MHC Class I molecule loaded with a peptide, an anti-CD3antibody, a superagonist anti-CD28 antibody, and a superagonist anti-CD2antibody.

The term “target cell” as used herein refers to cells which are involvedin a disease and can be targeted by the genetically modified cytotoxiccells of the invention (including but not limited to geneticallymodified T cells, NK cells, and macrophages). Other target cells will beapparent to those skilled in the art and may be used in connection withalternate embodiments of the invention.

The terms “T cell” and “T-lymphocyte” are interchangeable and usedsynonymously herein. Examples include, but are not limited to, naive Tcells, central memory T cells, effector memory T cells and combinationsthereof.

The term “transduction” as used herein refers to the introduction of aforeign nucleic acid into a cell using a viral vector. “Transfection” asused herein refers to the introduction of a foreign nucleic acid into acell using recombinant DNA technology. The term “transformation” meansthe introduction of a “foreign” (i.e. extrinsic or extracellular) gene,DNA or RNA sequence to a host cell, so that the host cell will expressthe introduced gene or sequence to produce a desired substance, such asa protein or enzyme coded by the introduced gene or sequence. Theintroduced gene or sequence may also be called a “cloned” or “foreign”gene or sequence, may include regulatory or control sequences, such asstart, stop, promoter, signal, secretion, or other sequences used by acell's genetic machinery. The gene or sequence may include nonfunctionalsequences or sequences with no known function. A host cell that receivesand expresses introduced DNA or RNA has been “transformed” and is a“transformant” or a “clone.” The DNA or RNA introduced to a host cellcan come from any source, including cells of the same genus or speciesas the host cell, or cells of a different genus or species

The term “treating” includes: (1) preventing or delaying the appearanceof clinical symptoms of the state, disorder or condition developing inan animal that may be afflicted with or predisposed to the state,disorder or condition but does not yet experience or display clinical orsubclinical symptoms of the state, disorder or condition; (2) inhibitingthe state, disorder or condition (i.e., arresting, reducing or delayingthe development of the disease, or a relapse thereof in case ofmaintenance treatment, of at least one clinical or subclinical symptomthereof); and/or (3) relieving the condition (i.e., causing regressionof the state, disorder or condition or at least one of its clinical orsubclinical symptoms). The benefit to a patient to be treated is eitherstatistically significant or at least perceptible to the patient or tothe physician.

“Tumor,” as used herein refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all pre-cancerous andcancerous cells and tissues

As used herein, the term “tumor microenvironment” refers to any and allelements of the tumor milieu including elements that create a structuraland or functional environment for the malignant process to surviveand/or expand and/or spread.

As used herein, the term “variable segment” refers to a portion of anascent peptide which includes a random, pseudorandom, or defined kernalsequence. A “variable segment” refers to a portion of a nascent peptidewhich includes a random pseudorandom, or defined kernal sequence. Avariable segment can include both variant and invariant residuepositions, and the degree of residue variation at a variant residueposition may be limited: both options are selected at the discretion ofthe practitioner. Typically, variable segments are about 5 to 20 aminoacid residues in length (e.g., 8 to 10), although variable segments maybe longer and may include antibody portions or receptor proteins, suchas an antibody fragment, a nucleic acid binding protein, a receptorprotein, and the like.

“Vector”, “cloning vector” and “expression vector” as used herein referto the vehicle by which a polynucleotide sequence (e.g. a foreign gene)can be introduced into a host cell, so as to transform the host andpromote expression (e.g. transcription and translation) of theintroduced sequence. Vectors include plasmids, phages, viruses, etc.

As used herein, the term “wild-type” means that the polynucleotide doesnot include any mutations. A “wild type protein”, “wild-type protein”,“wild-type biologic protein”, or “wild type biologic protein”, refers toa protein which can be isolated from nature that will be active at alevel of activity found in nature and will include the amino acidsequence found in nature. The terms “parent molecule” and “targetprotein” also refer to the wild-type protein. The “wild-type protein”preferably has some desired properties, such as higher binding affinity,or enzymatic activity, which may be obtained by screening of a libraryof proteins for a desired properties, including better stability indifferent temperature or pH environments, or improved selectivity and/orsolubility.

The term “working”, as in “working sample”, for example, is simply asample with which one is working. Likewise, a “working molecule”, forexample is a molecule with which one is working.

DETAILED DESCRIPTION

For illustrative purposes, the principles of the present invention aredescribed by referencing various exemplary embodiments. Although certainembodiments of the invention are specifically described herein, one ofordinary skill in the art will readily recognize that the sameprinciples are equally applicable to, and can be employed in othersystems and methods. Before explaining the disclosed embodiments of thepresent invention in detail, it is to be understood that the inventionis not limited in its application to the details of any particularembodiment shown. Additionally, the terminology used herein is for thepurpose of description and not of limitation. Furthermore, althoughcertain methods are described with reference to steps that are presentedherein in a certain order, in many instances, these steps may beperformed in any order as may be appreciated by one skilled in the art;the novel method is therefore not limited to the particular arrangementof steps disclosed herein.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural references unless thecontext clearly dictates otherwise. Furthermore, the terms “a” (or“an”), “one or more” and “at least one” can be used interchangeablyherein. The terms “comprising”, “including”, “having” and “constructedfrom” can also be used interchangeably.

The present disclosure is directed to a chimeric antigen receptor (CAR)for binding with a target antigen, comprising at least one antigenspecific targeting region evolved from a wild-type protein or a domainthereof and having at least one of: (a) a decrease in activity in theassay at the normal physiological condition compared to the antigenspecific targeting region of the wild-type protein or a domain thereof,and (b) an increase in activity in the assay under the aberrantcondition compared to the antigen specific targeting region of thewild-type protein or a domain thereof; a transmembrane domain; and anintracellular signaling domain. In some embodiments, the chimericantigen receptor further includes an extracellular spacer domain or atleast one co-stimulatory domain.

The CARs of the present invention have at least one of (1) theiraffinity to the target antigen reversibly or irreversibly reduced at thenormal physiological condition, and (2) an increased affinity, incomparison with the same CAR without the conditionally active antigenspecific targeting region. These CARs can direct cytotoxic cells to adisease site where an aberrant condition is present, such as a tumormicroenvironment or synovial fluid. As a result of these properties, theCARs can preferentially direct the cytotoxic cells to a disease sitewhile because of their low affinity for normal tissue. Such CARs candramatically reduce side-effects and allow higher doses of therapeuticsto be used to increase therapeutic efficacy. The CARs are particularlyvaluable for development of novel therapeutics that are required forshort or limited periods of time within a subject. Examples ofbeneficial applications include systemic treatments at high dosages, aswell as localized treatments at high concentrations.

The chimeric antigen receptor may include an antigen specific targetingregion that has a decrease in a binding affinity to the target antigenat a normal physiological condition compared to the antigen specifictargeting region of the wild-type protein or the domain thereof.

The chimeric antigen receptor many include an antigen specific targetingregion that has an increase in activity in the assay under the aberrantcondition compared to the antigen specific targeting region of thewild-type protein or a domain thereof and a decrease in a bindingaffinity to the target antigen at a normal physiological conditioncompared to the antigen specific targeting region of the wild-typeprotein or the domain thereof.

In any of the foregoing chimeric antigen receptors the antigen specifictargeting region may also have an increase in selectivity in the assayunder the aberrant condition compared to the antigen specific targetingregion of the wild-type protein or a domain thereof.

The CAR molecule includes a linker to connect the two antigen specifictargeting regions (FIG. 1). The linker orients the two antigen specifictargeting regions in such a way that the two antigen specific targetingregions on the CAR-T cells exhibit improved or optimal activity inbinding to the target antigen (Jensen et al., “Design and implementationof adoptive therapy with chimeric antigen receptor-modified T cells,”Immunol Rev., vol. 257, pp. 127-144, 2014). The linker is thuspreferably capable of adopting a specific conformation which enablesimproved or optimal binding of the two antigen specific targetingregions to the target antigen, thereby increasing the effectiveness ofthe CAR-T cells.

In some embodiments, the linker may be Gly-Ser tandem repeats in alength of 18-25 amino acids (Grada, “TanCAR: A Novel Bispecific ChimericAntigen Receptor for Cancer Immunotherapy,” Molecular Therapy NucleicAcids, vol. 2, e105, 2013). This flexible linker is capable of adoptingmany different conformations for improved or optimal presentation of twoantigen specific targeting regions for binding to the target antigen.

In some embodiments, the linker is capable of adopting differentconformations at a normal physiological condition and an aberrantcondition. Particularly, the linker has a first conformation at theaberrant condition which is improved or optimal for presentation of twoantigen specific targeting regions for binding to the target antigen,while the same linker has a second conformation at the normalphysiological condition which is less effective for presentation of twoantigen specific targeting regions for binding to the target antigenthan the first conformation of the linker under the aberrant condition.Such a linker may be called a “conditional linker” that allows the twoantigen specific targeting regions to bind to the target antigen at ahigher binding activity at an aberrant condition than at a normalphysiological condition. Therefore, CAR-T cells including such aconditional linker are more active at an aberrant condition than thesame CAR-T cells at a normal physiological condition.

Proteins that change conformation at different pH have been describedpreviously, for example, in Di Russo et al. (“pH-Dependentconformational changes in proteins and their effect on experimentalpK(a)s: the case of Nitrophorin 4,” PLoS Comput Biol., vol. 8, e1002761,2012). Further, proteins with different conformations at differenttemperatures have been described in Caldwell, “Temperature-inducedprotein conformational changes in barley root plasma membrane-enrichedmicrosomes,” Plant Physiol., vol. 84, pp. 924-929, 1989. Theconformation of antibodies being influenced by pH and/or temperature hasbeen discussed in Gandhi, “Effect of pH and temperature onconformational changes of a humanized monoclonal antibody,” Master'sthesis from University of Rhode Island, U.S.

It is within the scope of the present invention to select a conditionallinker to be used in the CAR molecule. The conditional linker can adopta first conformation at an aberrant condition, which is improved oroptimal for presenting the two antigen specific targeting regions forbinding to the target antigen, and adopt a second conformation at anormal physiological condition, which is suboptimal for presenting thetwo antigen specific targeting regions for binding to the targetantigen. In some embodiments, the suboptimal conformation of the linkerat the normal physiological condition produces a CAR molecule having abinding activity to the target antigen that is less than about 90%, orabout 80%, or about 70%, or about 60%, or about 50%, or about 40%, orabout 30%, or about 20%, or about 10%, or about 5% of the bindingactivity of the CAR molecule with the improved or optimal conformationof the linker at the aberrant condition.

The conditional linker may be generated from a starting linker selectedfrom 2A linkers, 2A-like linkers, picornaviral 2A-like linkers, a 2Apeptide of porcine teschovirus (P2A), and a 2A peptide of those a asignavirus (T2A), as well as variants and functional equivalents thereof. Thestarting linker is evolved to produce mutant proteins; the mutantproteins are then subjected to an assay at a normal physiologicalcondition and an assay at an aberrant condition. Proteins having aconditional linker are selected from the mutant proteins on the basisthat the selected proteins exhibit (a) a conditional linker having afirst conformation at the aberrant condition, which is improved oroptimal for presenting the two antigen specific targeting regions forbinding to the target antigen, and (b) a second conformation of theconditional linker at the normal physiological condition, which issuboptimal for presenting the two antigen specific targeting regions forbinding to the target antigen.

The CAR molecule also includes an extracellular spacer domain thatconnects the two antigen specific targeting regions with thetransmembrane domain, which, in turn, connects to the co-stimulatorydomain and the intracellular signaling domain inside of the T cells(FIG. 1). The extracellular spacer domain is preferably capable ofsupporting the antigen specific targeting regions to recognize and bindto the target antigen on the target cells (Hudecek et al., “Thenon-signaling extracellular spacer domain of chimeric antigen receptorsis decisive for in vivo antitumor activity,” Cancer Immunol Res., vol.3, pp. 125-135, 2015). In some embodiments, the extracellular spacerdomain is a flexible domain, thus allowing the antigen specifictargeting regions to have a structure to optimally recognize thespecific structure and density of the target antigens on a cell such astumor cell (Hudecek et al., “The non-signaling extracellular spacerdomain of chimeric antigen receptors is decisive for in vivo antitumoractivity,” Cancer Immunol Res., vol. 3, pp. 125-135, 2015). Theflexibility of the extracellular spacer domain permits the extracellularspacer domain to adopt many different conformations.

In some embodiments, the extracellular spacer domain is capable ofadopting different conformations at a normal physiological condition andan aberrant condition. Particularly, the extracellular spacer domain hasa first conformation at the aberrant condition which is improved oroptimal for presentation of two antigen specific targeting regions forbinding to the target antigen, while the same extracellular spacerdomain has a second conformation at the normal physiological conditionwhich is suboptimal for presentation of two antigen specific targetingregions for binding to the target antigen. Such an extracellular spacerdomain may be called a “conditional extracellular spacer domain” sinceit enables the two antigen specific targeting regions to bind to thetarget antigen at a higher binding activity at the aberrant conditionthan at the normal physiological condition. Therefore, with theconditional extracellular spacer domain, CAR-T cells may be more activeat the aberrant condition than the same CAR-T cells at the normalphysiological condition.

It is within the scope of the present invention to select a conditionalextracellular spacer domain to be used in the CAR molecule. In someembodiments, the suboptimal conformation of the extracellular spacerdomain at the normal physiological condition produces a CAR moleculehaving a binding activity to the target antigen that is less than about90%, or about 80%, or about 70%, or about 60%, or about 50%, or about40%, or about 30%, or about 20%, or about 10%, or about 5% of the CARmolecule with the optimal conformation for the same extracellular spacerdomain at the aberrant condition.

It has been discovered that the ubiquitylation-resistant form for theregion including the extracellular spacer domain and the transmembranedomain can enhance CAR-T cell signaling and thus augment antitumoractivity (Kunii et la, “Enhanced function of redirected human t cellsexpressing linker for activation of t cells that is resistant toubiquitylation,” Human Gene Therapy, vol. 24, pp. 27-37, 2013). Withinthis region, the extracellular spacer domain is outside of the CAR-Tcells, and thus is exposed to different conditions and can potentiallybe made conditionally ubiquitylation-resistant.

It is within the scope of the present invention that the extracellularspacer domain is conditionally ubiquitylation-resistant. Particularly,the extracellular spacer domain of the CAR molecule is moreubiquitylation-resistant at an aberrant condition than at a normalphysiological condition. Therefore, the CAR-T cells having theconditionally ubiquitylation-resistant extracellular spacer domain willhave enhanced cytotoxicity at the aberrant condition, relative to theircytotoxicity at the normal physiological condition.

The conditionally ubiquitylation-resistant extracellular spacer domainmay be selected to be more ubiquitylation-resistant at an aberrant pH oraberrant temperature, and less ubiquitylation-resistant at a normalphysiological pH or normal physiological temperature. In one embodiment,the conditionally ubiquitylation-resistant extracellular spacer domainis more ubiquitylation-resistant at a pH of a tumor microenvironment,and less ubiquitylation-resistant at a normal physiological pH, such asthe pH in human blood plasma at pH 7.2-7.6.

To produce a conditional extracellular spacer domain, a starting proteinfragment selected from an Fc fragment of an antibody, a hinge region ofan antibody, a CH2 region of an antibody, and a CH3 region of anantibody, is evolved to produce mutant proteins. The mutant proteins aresubjected to an assay at a normal physiological condition and an assayat an aberrant condition. The conditional extracellular spacer domain isselected from the mutant proteins that exhibit (a) a conditionalextracellular spacer domain that has a first conformation at theaberrant condition for the antigen specific targeting region to bind tothe target antigen at a higher binding activity and a secondconformation of the conditional extracellular binding domain at thenormal physiological condition for the antigen specific targeting regionto bind to the target antigen at a lower binding activity than at theaberrant condition, or proteins that are (b) moreubiquitylation-resistant at the aberrant condition than at the normalphysiological condition.

Any of the foregoing chimeric antigen receptors may be configured suchthat a protein containing the antigen receptor has an increase inexpression level compared to the wild-type protein or a domain thereof.

In an alternative embodiment, the present invention provides a chimericantigen receptor (CAR) for binding with a target antigen, including atleast one antigen specific targeting region evolved from a wild-typeprotein or a domain thereof and having an increase in selectivity in theassay under the aberrant condition compared to the antigen specifictargeting region of the wild-type protein or a domain thereof; atransmembrane domain; and an intracellular signaling domain. In someembodiments, the chimeric antigen receptor further includes anextracellular spacer domain or at least one co-stimulatory domain.

The present disclosure is also directed to methods of evolving awild-type protein or a domain thereof to generate a conditionally activeprotein that has at least one of: (a) a decrease in activity in theassay at the normal physiological condition compared to the antigenspecific targeting region of the wild-type protein or a domain thereof,and (b) an increase in activity in the assay under the aberrantcondition compared to the antigen specific targeting region of thewild-type protein or a domain thereof. The conditionally active proteinmay be engineered into a CAR.

The chimeric antigen receptor produced by the method may include anantigen specific targeting region that has a decrease in a bindingaffinity to the target antigen at a normal physiological conditioncompared to the antigen specific targeting region of the wild-typeprotein or the domain thereof.

The chimeric antigen receptor produced by the method may include anantigen specific targeting region that has an increase in activity inthe assay under the aberrant condition compared to the antigen specifictargeting region of the wild-type protein or a domain thereof and adecrease in a binding affinity to the target antigen at a normalphysiological condition compared to the antigen specific targetingregion of the wild-type protein or the domain thereof.

In any of the foregoing chimeric antigen receptors produced by themethod the antigen specific targeting region may also have an increasein selectivity in the assay under the aberrant condition compared to theantigen specific targeting region of the wild-type protein or a domainthereof.

Any of the foregoing chimeric antigen receptors produced by the methodmay be configured such that a protein containing the antigen receptorhas an increase in expression level compared to the wild-type protein ora domain thereof.

In an alternative embodiment of the method, the chimeric antigenreceptor (CAR) produced by the method for binding with a target antigen,includes at least one antigen specific targeting region evolved from awild-type protein or a domain thereof and having an increase inselectivity in the assay under the aberrant condition compared to theantigen specific targeting region of the wild-type protein or a domainthereof; a transmembrane domain; and an intracellular signaling domain.In some embodiments, the chimeric antigen receptor further includes anextracellular spacer domain or at least one co-stimulatory domain.

Chimeric Antigen Receptors

The immune system of mammals, especially humans, has cytotoxic cells fortargeting and destroying diseased tissue and/or pathogens. Using thesecytotoxic cells to remove unwanted tissue (i.e. target tissue) such astumors is a promising therapeutic approach. Other tissues that may betargeted for removal include glandular (e.g. prostate) hyperplasia,warts, and unwanted fatty tissue. However, this relatively newtherapeutic approach has achieved only limited success so far. Forexample, using T cells to target and destroy tumors has relatively lowlong term benefits because the cancer cells may adapted to the newtherapy by reducing expression of surface antigens to reduce theeffectiveness of this therapy. Cancer cells can even dedifferentiate toevade detection in response to tumor-specific T cells. See Maher,“Immunotherapy of Malignant Disease Using Chimeric Antigen ReceptorEngrafted T Cells,” ISRN Oncology, vol. 2012, article ID 278093, 2012.

Cytotoxic cells expressing chimeric antigen receptors can significantlyimprove the specificity and sensitivity of these cytotoxic cells. Forexample, T cells expressing a CAR (CAR-T cells) are capable of using theCAR to direct the T cells to target tumor cells expressing a cellsurface antigen that specifically binds to the CAR. Such CAR-T cells candeliver the cytotoxic agent more selectively to the tumor cells. CAR-Tcells can directly recognize a target molecule and thus are typicallynot restricted by polymorphic presenting elements such as humanleukocyte antigens (HLAs). Advantages of this CAR targeting strategy arethreefold. First, since the CAR-T cell function is not dependent uponHLA status, the same CAR-based approach can in principle be used in allpatients with tumors that express the same target surface antigen.Second, corruption of antigen processing and presenting machinery is acommon attribute of tumor cells and may facilitate immune escape.However, this affords no protection against CAR-T cells. Third, a rangeof macromolecules can be targeted using this system, including proteins,carbohydrates, and glycolipids.

A chimeric antigen receptor of the present invention is a chimericartificial protein including at least one antigen specific targetingregion (ASTR), a transmembrane domain (TM), and an intracellularsignaling domain (ISD). In some embodiments, the CAR may further includean extracellular spacer domain (ESD) and/or a co-stimulatory domain(CSD). See FIG. 1.

The ASTR is an extracellular region of the CAR for binding to a specifictarget antigen including proteins, carbohydrates, and glycolipids. Insome embodiments, the ASTR includes an antibody, especially asingle-chain antibody, or a fragment thereof. The ASTR may include afull length heavy chain, an Fab fragment, a single chain Fv (scFv)fragment, a divalent single chain antibody or a diabody, each of whichare specific to the target antigen.

The ASTR may also include another protein functional domain to recognizeand bind to the target antigen. Because the target antigen may haveother biological functions, such as acting as a receptor or a ligand,the ASTR may alternatively include a functional domain for specificallybinding with the antigen. Some examples of proteins with functionaldomains include linked cytokines (which leads to recognition of cellsbearing the cytokine receptor), affibodies, ligand binding domains fromnaturally occurring receptors, soluble protein/peptide ligands for areceptor, for example on a tumor cell. In fact, almost any molecule thatis capable of binding to a given antigen with high affinity can be usedin the ASTR, as will be appreciated by those skilled in the art.

In one embodiment, the CAR of the invention includes at least two ASTRswhich target at least two different antigens or two epitopes on the sameantigen. In an embodiment, the CAR includes three or more ASTRs whichtarget at least three or more different antigens or epitopes. When aplurality of ASTRs is present in the CAR, the ASTRs may be arranged intandem and may be separated by linker peptides (FIG. 1).

In one embodiment, the ASTR includes a full-length IgG heavy chain thatis specific for the target antigen and having the V_(H), CH1, hinge, andthe CH2 and CH3 (Fc) Ig domains, if the V_(H) domain alone is sufficientto confer antigen-specificity (“single-domain antibodies”). If both, theV_(H) and the V_(L) domains are necessary to generate a fully activeASTR, the V_(H)-containing CAR and the full-length lambda light chain(IgL) are both introduced into the same cytotoxic cell to generate anactive ASTR. In another embodiment, each ASTR of the CAR includes atleast two single chain antibody variable fragments (scFv), each specificfor a different target antigen. scFvs, in which the C-terminus of onevariable domain (V_(H) or V_(L)) is tethered to the N-terminus of theother variable domain (V_(L) or V_(H), respectively) via a polypeptidelinker, have been developed without significantly disrupting antigenbinding or specificity of the binding (Chaudhary et al., “A recombinantsingle-chain immunotoxin composed of anti-Tac variable regions and atruncated diphtheria toxin,” Proc. Natl. Acad. Sci., vol. 87, page 9491,1990; Bedzyk et al.,” “Immunological and structural characterization ofa high affinity anti-fluorescein single-chain antibody,” J. Biol. Chem.,vol. 265, page 18615, 1990). These scFvs lack the constant regions (Fc)present in the heavy and light chains of a native antibody. The scFvs,specific for at least two different antigens, are arranged in tandem. Inan embodiment, an extracelluar spacer domain may be linked between theASTR and the transmembrane domain.

In another embodiment, an scFv fragment may be fused to all or a portionof the constant domains of the heavy chain. In a further embodiment, anASTR of the CAR includes a divalent (or bivalent) single-chain variablefragment (di-scFvs, bi-scFvs). In CARs including di-scFVs, two scFvseach specific for an antigen are linked together to form a singlepeptide chain with two V_(H) and two V_(L) regions (Xiong et al.,“Development of tumor targeting anti-MUC-1 multimer: effects of di-scFvunpaired cysteine location on PEGylation and tumor binding,” ProteinEngineering Design and Selection, vol. 19, pages 359-367, 2006; Kufer etal., “A revival of bispecific antibodies,” Trends in Biotechnology, vol.22, pages 238-244, 2004).

In yet another embodiment, an ASTR includes a diabody. In a diabody, thescFvs are created with linker peptides that are too short for the twovariable regions to fold together, driving the scFvs to dimerize. Stillshorter linkers (one or two amino acids) lead to the formation oftrimers, the so-called triabodies or tribodies. Tetrabodies may also beused in the ASTR.

When two or more ASTRs are present in a CAR, the ASTRs are connected toeach other covalently on a single polypeptide chain, through an oligo-or polypeptide linker, an Fc hinge or a membrane hinge region.

The antigens targeted by the CAR are present on the surface or inside ofcells in a tissue that targeted for removal, such as tumors, glandular(e.g. prostate) hyperplasia, warts, and unwanted fatty tissue. While thesurface antigens are more efficiently recognized and bound by the ASTRof CARs, intracellular antigens may also be targeted by the CARs. Insome embodiments, the target antigens are preferably specific forcancer, inflammatory disease, neuronal-disorders, diabetes,cardiovascular disease, or infectious diseases. Examples of targetantigens include antigens expressed by various immune cells, carcinomas,sarcomas, lymphomas, leukemia, germ cell tumors, blastomas, and cellsassociated with various hematologic diseases, autoimmune diseases,and/or inflammatory diseases.

Antigens specific for cancer which may be targeted by the ASTR includeone or more of 4-IBB, 5T4, adenocarcinoma antigen, alpha-fetoprotein,BAFF, B-lymphoma cell, C242 antigen, CA-125, carbonic anhydrase 9(CA-IX), C-MET, CCR4, CD152, CD19, CD20, CD200, CD22, CD221, CD23 (IgEreceptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44 v6, CD51, CD52,CD56, CD74, CD80, CEA, CNT0888, CTLA-4, DRS, EGFR, EpCAM, CD3, FAP,fibronectin extra domain-B, folate receptor 1, GD2, GD3 ganglioside,glycoprotein 75, GPNMB, HER2/neu, HGF, human scatter factor receptorkinase, IGF-1 receptor, IGF-I, IgG1, LI-CAM, IL-13, IL-6, insulin-likegrowth factor I receptor, integrin α5β1, integrin αvβ3, MORAb-009,MS4A1, MUC1, mucin CanAg, N-glycolylneuraminic acid, NPC-1C, PDGF-R a,PDL192, phosphatidylserine, prostatic carcinoma cells, RANKL, RON, ROR1,SCH 900105, SDC1, SLAMF7, TAG-72, tenascin C, TGF beta 2, TGF-β,TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2 orvimentin.

Antigens specific for inflammatory diseases which may be targeted by theASTR include one or more of AOC3 (VAP-1), CAM-3001, CCL11 (eotaxin-1),CD125, CD147 (basigin), CD154 (CD40L), CD2, CD20, CD23 (IgE receptor),CD25 (a chain of IL-2 receptor), CD3, CD4, CD5, IFN-α, IFN-γ, IgE, IgEFc region, IL-1, IL-12, IL-23, IL-13, IL-17, IL-17A, IL-22, IL-4, IL-5,IL-5, IL-6, IL-6 receptor, integrin a4, integrin α4β7, Lama glama, LFA-1(CD1 la), MEDI-528, myostatin, OX-40, rhuMAb 137, scleroscin, SOST, TGFbeta 1, TNF-α or VEGF-A.

Antigens specific for neuronal disorders which may be targeted by theASTR of the invention include one or more of beta amyloid or MABT5102A.Antigens specific for diabetes which may be targeted by the ASTR of theinvention include one or more of L-Iβ or CD3. Antigens specific forcardiovascular diseases which may be targeted by the ASTR of theinvention include one or more of C5, cardiac myosin, CD41 (integrinalpha-lib), fibrin II, beta chain, ITGB2 (CD 18) andsphingosine-1-phosphate.

Antigens specific for infectious diseases which may be targeted by theASTR of the invention include one or more of anthrax toxin, CCR5, CD4,clumping factor A, cytomegalovirus, cytomegalovirus glycoprotein B,endotoxin, Escherichia coli, hepatitis B surface antigen, hepatitis Bvirus, HIV-1, Hsp90, Influenza A hemagglutinin, lipoteichoic acid,Pseudomonas aeruginosa, rabies virus glycoprotein, respiratory syncytialvirus and TNF-α.

Further examples of target antigens include surface proteins found oncancer cells in a specific or amplified fashion, e.g. the IL-14receptor, CD19, CD20 and CD40 for B-cell lymphoma, the Lewis Y and CEAantigens for a variety of carcinomas, the Tag72 antigen for breast andcolorectal cancer, EGF-R for lung cancer, folate binding protein and theHER-2 protein which is often amplified in human breast and ovariancarcinomas, or viral proteins, e.g. gp120 and gp41 envelope proteins ofHIV, envelope proteins from the Hepatitis B and C viruses, glycoproteinB and other envelope glycoproteins of human cytomegalovirus, and theenvelope proteins from oncoviruses such as Kaposi's sarcoma-associatedHerpes virus. Other potential target antigens include CD4, where theligand is the HIV gp120 envelope glycoprotein, and other viralreceptors, for example ICAM, which is the receptor for the humanrhinovirus, and the related receptor molecule for poliovirus.

In another embodiment, the CAR may target antigens that engagecancer-treating cells, such as NK cells and other cells mentionedherein, to activate the cancert-treating cells by acting as immuneeffector cells. One example of this is a CAR that targets the CD16Aantigen to engage NK cells to fight CD30-expressing malignancies. Thebispecific, tetravalent AFM13 antibody is an example of an antibody thatcan deliver this effect. Further details of this type of embodiment canbe found, for example, in Rothe, A., et al., “A phase 1 study of thebispecific anti-CD30/CD16A antibody construct AFM13 in patients withrelapsed or refractory Hodgkin lymphoma,” Blood, 25 Jun. 2015, Vl. 125,no. 26, pp. 4024-4031.

The extracellular spacer domain of the CAR is a hydrophilic region whichis located between the ASTR and the transmembrane domain. In someembodiments, this domain facilitates proper protein folding for the CAR.The extracellular spacer domain is an optional component for the CAR.The extracellular spacer domain may include a domain selected from Fcfragments of antibodies, hinge regions of antibodies, CH2 regions ofantibodies, CH3 regions of antibodies, artificial spacer sequences orcombinations thereof. Examples of extracellular spacer domains includeCD8a hinge, artificial spacers made of polypeptides which may be assmall as, three glycines (Gly), as well as CH1 and CH3 domains of IgGs(such as human IgG4).

The transmembrane domain of the CAR is a region that is capable ofspanning the plasma membrane of the cytotoxic cells. The transmembranedomain is selected from a transmembrane region of a transmembraneprotein such as, for example, Type I transmembrane proteins, anartificial hydrophobic sequence or a combination thereof. Examples ofthe transmembrane domain include the transmembrane regions of the alpha,beta or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4,CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137,CD154. Synthetic transmembrane domains may include a triplet ofphenylalanine, tryptophan and valine. Optionally, a short oligo- orpolypeptide linker, preferably between 2 and 10 amino acids in length,may form the linkage between the transmembrane domain and theintracellular signaling domain of the CAR. A glycine-serine doubletprovides a particularly suitable linker between the transmembrane domainand the intracellular signaling domain.

The CAR of the invention also includes an intracellular signalingdomain. The intracellular signaling domain transduces the effectorfunction signal and directs the cytotoxic cell to perform itsspecialized function, i.e., harming and/or destroying the target cells.Examples of the intracellular signaling domain include the ζ chain ofthe T cell receptor complex or any of its homologs, e.g., η chain,FcsRly and β chains, MB 1 (Iga) chain, B29 (Ig) chain, etc., human CD3zeta chain, CD3 polypeptides (Δ, δ and ε), syk family tyrosine kinases(Syk, ZAP 70, etc.), src family tyrosine kinases (Lck, Fyn, Lyn, etc.)and other molecules involved in T cell transduction, such as CD2, CD5and CD28. Specifically, the intracellular signaling domain may be humanCD3 zeta chain, FcyRIII, FcsRI, cytoplasmic tails of Fc receptors, animmunoreceptor tyrosine-based activation motif (ITAM) bearingcytoplasmic receptors and combinations thereof.

The intracellular signaling domains used in the CAR may includeintracellular signaling domains of several types of various other immunesignaling receptors, including, but not limited to, first, second, andthird generation T cell signaling proteins including CD3, B7 familycostimulatory, and Tumor Necrosis Factor Receptor (TNFR) superfamilyreceptors (Park et al., “Are all chimeric antigen receptors createdequal?” J Clin Oncol., vol. 33, pp. 651-653, 2015). Additionallyintracellular signaling domains include signaling domains used by NK andNKT cells (Hermanson, et al., “Utilizing chimeric antigen receptors todirect natural killer cell activity,” Front Immunol., vol. 6, p. 195,2015) such as signaling domains of NKp30 (B7-H6) (Zhang et al., “AnNKp30-based chimeric antigen receptor promotes T cell effector functionsand antitumor efficacy in vivo,” J Immunol., vol. 189, pp. 2290-2299,2012), and DAP12 (Topfer et al., “DAP12-based activating chimericantigen receptor for NK cell tumor immunotherapy,” J Immunol., vol. 194,pp. 3201-3212, 2015), NKG2D, NKp44, NKp46, DAP10, and CD3z. Additionallyintracellular signaling domains also includes signaling domains of humanImmunoglobulin receptors that contain immunoreceptor tyrosine basedactivation motif (ITAM) such as FcgammaRI, FcgammaRIIA, FcgammaRIIC,FcgammaRIIIA, FcRL5 (Gillis et al., “Contribution of Human FcγRs toDisease with Evidence from Human Polymorphisms and Transgenic AnimalStudies,” Front Immunol., vol. 5, p. 254, 2014).

In some embodiments, the intracellular signaling domain includes acytoplasmic signaling domain of TCR zeta, FcR gamma, FcR beta, CD3gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, or CD66d. It isparticularly preferred that the intracellular signaling domain in theCAR includes a cytoplasmic signaling domain of human CD3 zeta.

The CAR of the present invention may include a co-stimulatory domain,which has the function of enhancing cell proliferation, cell survivaland development of memory cells for the cytotoxic cells that express theCAR. The CAR of the invention may include one or more co-stimulatorydomains selected from co-stimulatory domains of proteins in the TNFRsuperfamily, CD28, CD137 (4-1BB), CD134 (OX40), DaplO, CD27, CD2, CD7,CD5, ICAM-1, LFA-1(CD1 la/CD18), Lck, TNFR-I, PD-1, TNFR-II, Fas, CD30,CD40, ICOS LIGHT, NKG2C, B7-H3, or combinations thereof. If the CARincludes more than one co-stimulatory domain, these domains may bearranged in tandem, optionally separated by a linker. The co-stimulatorydomain is an intracellular domain that may locate between thetransmembrane domain and the intracellular signaling domain in the CAR.

In some embodiments, two or more components of the CAR of the inventionare separated by one or more linkers. For example, in a CAR including atleast two ASTRs, the two ASTRs may be separated by a linker. Linkers areoligo- or polypeptide regions of from about 1 to 100 amino acids inlength. In some embodiments, the linkers may be, for example, 5-12 aminoacids in length, 5-15 amino acids in length or 5 to 20 amino acids inlength. Linkers may be composed of flexible residues like glycine andserine so that the adjacent protein domains are free to move relative toone another. Longer linkers, for example those longer than 100 aminoacids, may be used in connection with alternate embodiments of theinvention, and may be selected to, for example, ensure that two adjacentdomains do not sterically interfere with one another. Examples oflinkers which may be used in the instant invention include but are notlimited to 2A linkers (for example T2A), 2A-like linkers or functionalequivalents thereof.

Conditionally Active Antigen Specific Targeting Region

The CARs are chimeric proteins that are generated by fusing all thedifferent domains discussed above together to form a fusion protein. TheCAR is typically generated by an expression vector includingpolynucleotide sequences that encode the different domains of the CAR.The ASTR of the present invention, which functions to recognize and bindwith an antigen on target cells, is conditionally active. Specifically,the ASTR is less active or inactive at a normal physiological conditionand active at an aberrant condition for binding with the target antigen,in comparison with an ASTR of the corresponding wild-type protein. Thepresent invention provides a method to generate the conditionally activeASTR from a wild-type protein or its binding domain (wild-type ASTR).

The wild-type protein that suitable to be used in whole or in part forat least its binding domain for the target antigen, as an ASTR in thepresent invention may be discovered by generating a protein library andscreening the library for a protein with a desired binding affinity tothe target antigen. The wild-type protein may be discovered by screeninga cDNA library. A cDNA library is a combination of cloned cDNA(complementary DNA) fragments inserted into a collection of host cells,which together constitute some portion of the transcriptome of theorganism. cDNA is produced from fully transcribed mRNA and thereforecontains the coding sequence for expressed proteins of an organism. Theinformation in cDNA libraries is a powerful and useful tool fordiscovery of proteins with desired properties by screening the librariesfor proteins with the desired binding affinity to the target antigen.

In some embodiments where the wild-type proteins are antibodies, thewild-type antibodies can be discovered by generating and screeningantibody libraries. The antibody libraries can be either polyclonalantibody libraries or monoclonal antibody libraries. A polyclonalantibody library against a target antigen can be generated by directinjection of the antigen into an animal or by administering the antigento a non-human animal. The antibodies so obtained represent a library ofpolyclonal antibodies that bind to the antigen. For preparation ofmonoclonal antibody libraries, any technique which provides antibodiesproduced by continuous cell line cultures can be used. Examples includethe hybridoma technique, the trioma technique, the human B-cellhybridoma technique, and the EBV-hybridoma technique (see, e.g., Cole(1985) in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc.,pp. 77-96). Techniques described for the generating single chainantibodies (see, e.g., U.S. Pat. No. 4,946,778) can be adapted toproduce single chain antibody library.

There are other methods for generation and screening of antibodylibraries for discovery of the wild-type antibody. For example, fullyhuman antibody display libraries can be utilized. Such a library is apopulation of antibodies displayed on the surface of host cell(s).Preferably, the antibody library is representative of the humanrepertoire of antibodies in that they have the capability of binding toa wide range of antigens. Because the antibodies are displayed on thesurface of cells, the effective affinity (due to avidity) of eachantibody in the library is increased. Unlike other popular librarytypes, such as phage display libraries, where avidity of the antibodiesfor screening and identification purposes is less desirable, the superavidity provided by cell surface display in the present invention, isdesirable. Cell surface display libraries enable the identification oflow, medium and high binding affinity antibodies, as well as theidentification of non-immunogenic and weak epitopes in the screening orselection step.

Generation of Evolved Molecules from Parent Molecule

The wild-type protein, or its binding domain (wild-type ASTR) undergoesa process of mutagenesis to produce a population of mutant polypeptides,which can then be screened to identify a mutant ASTR with an enhancedbinding affinity to the target antigen at an aberrant condition, andoptionally, substantially the same or a reduction in binding affinity tothe target antigen at a normal physiological condition, in comparisonwith the wild-type ASTR.

Any chemical synthetic or recombinant mutagenic method may be used togenerate the population of mutant polypeptides. The practice of thepresent invention may employ, unless otherwise indicated, conventionaltechniques of cell biology, cell culture, molecular biology, transgenicbiology, microbiology, recombinant DNA, and immunology, which are withinthe skill of the art. Such techniques are explained fully in theliterature. See, for example, Molecular Cloning A Laboratory Manual, 2ndEd., ed. by Sambrook, Fritsch and Maniatis (Cold Spring HarborLaboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glovered., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis etal. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D. Hames &S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames &S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, AlanR. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986);B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise,Methods In Enzymology (Academic Press, Inc., N.Y.); Gene TransferVectors For Mammalian Cells (J. H. Miller and M. P. Cabs eds., 1987,Cold Spring Harbor Laboratory); Methods In Enzymnology, Vols. 154 and155 (Wu et al. eds.), Immunochemical Methods In Cell And MolecularBiology (Mayer and Walker, eds., Academic Press, London, 1987); HandbookOf Experimental Immunology, Volumes 1-IV (D. M. Weir and C. C.Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

The disclosure provides for a method for generating a nucleic acidmutant encoding a mutant polypeptide being conditionally active, themethod including modifying the nucleic acid by (i) substituting one ormore nucleotides for a different nucleotide, wherein the nucleotideincludes a natural or non-natural nucleotide; (ii) deleting one or morenucleotides, (iii) adding one or more nucleotides, or (iv) anycombination thereof. In one aspect, the non-natural nucleotide includesinosine. In another aspect, the method further includes assaying thepolypeptides encoded by the modified nucleic acids for altered enzymeactivity, thereby identifying the modified nucleic acid(s) encoding apolypeptide having altered enzyme activity. In one aspect, themodifications of step (a) are made by PCR, error-prone PCR, shuffling,oligonucleotide-directed mutagenesis, assembly PCR, sexual PCRmutagenesis, in vivo mutagenesis, cassette mutagenesis, recursiveensemble mutagenesis, exponential ensemble mutagenesis, site-specificmutagenesis, gene reassembly, gene site saturated mutagenesis, ligasechain reaction, in vitro mutagenesis, ligase chain reaction,oligonuclteotide synthesis, any DNA-generating technique and anycombination thereof. In another aspect, the method further includes atleast one repetition of the modifying step.

The disclosure further provides a method for making a polynucleotidefrom two or more nucleic acids, the method including: (a) identifyingregions of identity and regions of diversity between two or more nucleicacids, wherein at least one of the nucleic acids includes a nucleic acidof the disclosure; (b) providing a set of oligonucleotides whichcorrespond in sequence to at least two of the two or more nucleic acids;and, (c) extending the oligonucleotides with a polymerase, therebymaking the polynucleotide.

Any technique of mutagenesis can be employed in various embodiments ofthe disclosure. Stochastic or random mutagenesis is exemplified by asituation in which a parent molecule is mutated (modified or changed) toyield a set of progeny molecules having mutation(s) that are notpredetermined. Thus, in an in vitro stochastic mutagenesis reaction, forexample, there is not a particular predetermined product whoseproduction is intended; rather there is an uncertainty—hencerandomness—regarding the exact nature of the mutations achieved, andthus also regarding the products generated. Stochastic mutagenesis ismanifested in processes such as error-prone PCR and stochasticshuffling, where the mutation(s) achieved are random or notpredetermined. The variant forms can be generated by error-pronetranscription, such as an error-prone PCR or use of a polymerase whichlacks proof-reading activity (see, Liao (1990) Gene 88: 107-111), of thefirst variant form, or, by replication of the first form in a mutatorstrain (mutator host cells are discussed in further detail below, andare generally well known). A mutator strain can include any mutants inany organism impaired in the functions of mismatch repair. These includemutant gene products of mutS, mutT, mutH, mutL, ovrD, dcm, vsr, umuC,umuD, sbcB, recJ, etc. The impairment is achieved by genetic mutation,allelic replacement, selective inhibition by an added reagent such as asmall compound or an expressed antisense RNA, or other techniques.Impairment can be of the genes noted, or of homologous genes in anyorganism.

Other mutagenesis methods include oligonucleotide-directed mutagenesistechnologies, error-prone polymerase chain reactions (error-prone PCR)and cassette mutagenesis, in which a specific region of the parentalpolynucleotide is replaced with a synthetically mutagenizedoligonucleotide. In these cases, a number of mutant sites are generatedaround certain sites in the parental sequence.

In oligonucleotide-directed mutagenesis, a short sequence is replacedwith a synthetically mutagenized oligonucleotide. Inoligonucleotide-directed mutagenesis, a short sequence of thepolynucleotide is removed from the polynucleotide using restrictionenzyme digestion and is replaced with a synthetic polynucleotide inwhich various bases have been altered from the original sequence. Thepolynucleotide sequence can also be altered by chemical mutagenesis.Chemical mutagens include, for example, sodium bisulfite, nitrous acid,hydroxylamine, hydrazine or formic acid. Other agents which areanalogues of nucleotide precursors include nitrosoguanidine,5-bromouracil, 2-aminopurine, or acridine. Generally, these agents areadded to the PCR reaction in place of the nucleotide precursor therebymutating the sequence. Intercalating agents such as proflavine,acriflavine, quinacrine and the like can also be used. Randommutagenesis of the polynucleotide sequence can also be achieved byirradiation with X-rays or ultraviolet light. Generally, plasmidpolynucleotides so mutagenized are introduced into E. coli andpropagated as a pool or library of hybrid plasmids.

Error-prone PCR uses low-fidelity polymerization conditions to introducea low level of point mutations randomly over a long sequence. In amixture of fragments of unknown sequence, error-prone PCR can be used tomutagenize the mixture.

In cassette mutagenesis, a sequence block of a single template istypically replaced by a (partially) randomized sequence. Reidhaar-OlsonJ F and Sauer R T: Combinatorial cassette mutagenesis as a probe of theinformational content of protein sequences. Science 241(4861):53-57,1988.

Alternatively, any technique of non-stochastic or non-random mutagenesiscan be employed in various embodiments of the disclosure. Non-stochasticmutagenesis is exemplified by a situation in which a parent molecule ismutated (modified or changed) to yield a progeny molecule having one ormore predetermined mutations. It is appreciated that the presence ofbackground products in some quantity is a reality in many reactionswhere molecular processing occurs, and the presence of these backgroundproducts does not detract from the non-stochastic nature of amutagenesis process having a predetermined product. Site-saturationmutagenesis and synthetic ligation reassembly, are examples ofmutagenesis techniques where the exact chemical structure(s) of theintended product(s) are predetermined.

One method of site-saturation mutagenesis is disclosed in U.S. patentapplication publication 2009/0130718 This method provides a set ofdegenerate primers corresponding to codons of a template polynucleotide,and performs polymerase elongation to produce progeny polynucleotides,which contain sequences corresponding to the degenerate primers. Theprogeny polynucleotides can be expressed and screened for directedevolution. Specifically, this is a method for producing a set of progenypolynucleotides, including the steps of (a) providing copies of atemplate polynucleotide, each including a plurality of codons thatencode a template polypeptide sequence; and (b) for each codon of thetemplate polynucleotide, performing the steps of (1) providing a set ofdegenerate primers, where each primer includes a degenerate codoncorresponding to the codon of the template polynucleotide and at leastone adjacent sequence that is homologous to a sequence adjacent to thecodon of the template polynucleotide; (2) providing conditions allowingthe primers to anneal to the copies of the template polynucleotides; and(3) performing a polymerase elongation reaction from the primers alongthe template; thereby producing progeny polynucleotides, each of whichcontains a sequence corresponding to the degenerate codon of theannealed primer; thereby producing a set of progeny polynucleotides.Site-saturation mutagenesis relates to the directed evolution of nucleicacids and screening of clones containing the evolved nucleic acids forresultant binding activity of interest.

Site saturation mutagenesis relates generally to a method of: 1)preparing a progeny generation of molecule(s) (including a molecule thatis included of a polynucleotide sequence, a molecule that is included ofa polypeptide sequence, and a molecule that is included in part of apolynucleotide sequence and in part of a polypeptide sequence), that ismutagenized to achieve at least one point mutation, addition, deletion,and/or chimerization, from one or more ancestral or parental generationtemplate(s); 2) screening the progeny generation molecule(s)—preferablyusing a high throughput method—for desired binding affinity to thetarget antigen; 3) optionally obtaining &/or cataloguing structural &/orand functional information regarding the parental &/or progenygeneration molecules; and 4) optionally repeating any of steps 1) to 3).

In site saturation mutagenesis, there is generated (e.g. from a parentpolynucleotide template)-in what is termed “codon site-saturationmutagenesis”—a progeny generation of polynucleotides, each having atleast one set of up to three contiguous point mutations (i.e. differentbases including a new codon), such that every codon (or every family ofdegenerate codons encoding the same amino acid) is represented at eachcodon position. Corresponding to—and encoded by—this progeny generationof polynucleotides, there is also generated a set of progenypolypeptides, each having at least one single amino acid point mutation.In a preferred aspect, there is generated—in what is termed “amino acidsite-saturation mutagenesis”-one such mutant polypeptide for each of the19 naturally encoded polypeptide-forming alpha-amino acid substitutionsat each and every amino acid position along the polypeptide. Thisyields—for each and every amino acid position along the parentalpolypeptide—a total of 20 distinct progeny polypeptides including theoriginal amino acid, or potentially more than 21 distinct progenypolypeptides if additional amino acids are used either instead of or inaddition to the 20 naturally encoded amino acids.

Other mutagenesis techniques can also be employed which involverecombination and more specifically a method for preparingpolynucleotides encoding a polypeptide by a method of in vivore-assortment of polynucleotide sequences containing regions of partialhomology, assembling the polynucleotides to form at least onepolynucleotide and screening the polynucleotides for the production ofpolypeptide(s) having a useful property.

In another aspect, mutagenesis techniques exploit the natural propertyof cells to recombine molecules and/or to mediate reductive processesthat reduce the complexity of sequences and extent of repeated orconsecutive sequences possessing regions of homology.

Various mutagenesis techniques can be used alone or in combination toprovide a method for generating hybrid polynucleotides encodingbiologically active hybrid polypeptides. In accomplishing these andother objects, there has been provided, in accordance with one aspect ofthe disclosure, a method for introducing polynucleotides into a suitablehost cell and growing the host cell under conditions that produce hybridpolypeptides.

Chimeric genes have been made by joining 2 polynucleotide fragmentsusing compatible sticky ends generated by restriction enzyme(s), whereeach fragment is derived from a separate progenitor (or parental)molecule. Another example is the mutagenesis of a single codon position(i.e. to achieve a codon substitution, addition, or deletion) in aparental polynucleotide to generate a single progeny polynucleotideencoding for a single site-mutagenized polypeptide.

Further, in vivo site specific recombination systems have been utilizedto generate hybrids of genes, as well as random methods of in vivorecombination, and recombination between homologous but truncated geneson a plasmid. Mutagenesis has also been reported by overlappingextension and PCR.

Non-random methods have been used to achieve larger numbers of pointmutations and/or chimerizations, for example comprehensive or exhaustiveapproaches have been used to generate all the molecular species within aparticular grouping of mutations, for attributing functionality tospecific structural groups in a template molecule (e.g. a specificsingle amino acid position or a sequence included of two or more aminoacids positions), and for categorizing and comparing specific groupingof mutations.

Any of these or other methods of evolving can be employed in the presentdisclosure to generate a new population of mutant polypeptides (library)from the wild-type protein.

Expression of Evolved Molecules

The mutant polynucleotides generated from the evolving step may, or maynot be size fractionated on an agarose gel according to publishedprotocols, inserted into an expression vector, and transfected into anappropriate host cell to produce the mutant polypeptides (expression).The expression may use routine molecular biology techniques. Thus, theexpression step can use various known methods.

For example, briefly, mutant polynucleotides generated from the evolvingstep are then digested and ligated into an expression vector, such asplasmid DNA using standard molecular biology techniques. The vector isthen transformed into bacteria or other cells using standard protocols.This can be done in an individual well of a multi-well tray, such as a96-well tray for high throughput expression and screening. The processis repeated for each mutant polynucleotide.

Polynucleotides selected and isolated as described are introduced into asuitable host cell. A suitable host cell is any cell which is capable ofpromoting recombination and/or reductive reassortment. The selectedpolynucleotides are preferably already in a vector which includesappropriate control sequences. The host cell can be a higher eukaryoticcell, such as a mammalian cell, or a lower eukaryotic cell, such as ayeast cell, or preferably, the host cell can be a prokaryotic cell, suchas a bacterial cell. Introduction of the construct into the host cellcan be effected by calcium phosphate transfection, DEAE-Dextran mediatedtransfection, or electroporation (e.g. Ecker and Davis, 1986, Inhibitionof gene expression in plant cells by expression of antisense RNA, ProcNatl Acad Sci USA, 83:5372-5376).

As representative examples of expression vectors which may be used,there may be mentioned viral particles, baculovirus, phage, plasmids,phagemids, cosmids, fosmids, bacterial artificial chromosomes, viral DNA(e.g., vaccinia, adenovirus, foul pox virus, pseudorabies andderivatives of SV40), Pl-based artificial chromosomes, yeast plasmids,yeast artificial chromosomes, and any other vectors specific forspecific hosts of interest (such as bacillus, aspergillus and yeast).Thus, for example, the DNA may be included in any one of a variety ofexpression vectors for expressing a polypeptide. Such vectors includechromosomal, nonchromosomal and synthetic DNA sequences. Large numbersof suitable vectors are known to those of skill in the art, and arecommercially available. The following vectors are provided by way ofexample; Bacterial: pQE vectors (Qiagen), pBluescript plasmids, pNHvectors, (lambda-ZAP vectors (Stratagene); ptrc99a, pKK223-3, pDR540,pRIT2T (Pharmacia); Eukaryotic: pXTl, ρSG5 (Stratagene), pSVK3, pBPV,pMSG, pSVLSV40 (Pharmacia). However, any other plasmid or other vectormay be used so long as they are replicable and viable in the host. Lowcopy number or high copy number vectors may be employed with the presentdisclosure.

The mutant polynucleotide sequence in the expression vector isoperatively linked to an appropriate expression control sequence(s)(promoter) to direct RNA synthesis. Particular named bacterial promotersinclude lad, lacZ, T3, T7, gpt, lambda PR, PL and trp. Eukaryoticpromoters include CMV immediate early, HSV thymidine kinase, early andlate SV40, LTRs from retrovirus, and mouse metallothionein-1. Selectionof the appropriate vector and promoter is well within the level ofordinary skill in the art. The expression vector also contains aribosome binding site for translation initiation and a transcriptionterminator. The vector may also include appropriate sequences foramplifying expression. Promoter regions can be selected from any desiredgene using chloramphenicol transferase (CAT) vectors or other vectorswith selectable markers. In addition, the expression vectors preferablycontain one or more selectable marker genes to provide a phenotypictrait for selection of transformed host cells such as dihydrofolatereductase or neomycin resistance for eukaryotic cell culture, or such astetracycline or ampicillin resistance in E. coli.

Eukaryotic DNA transcription can be increased by inserting an enhancersequence into the expression vector Enhancers are cis-acting sequencesof between 10 to 300 bp that increase transcription by a promoter.Enhancers can effectively increase transcription when either 5′ or 3′ tothe transcription unit. They are also effective if located within anintron or within the coding sequence itself. Typically, viral enhancersare used, including SV40 enhancers, cytomegalovirus enhancers, polyomaenhancers, and adenovirus enhancers. Enhancer sequences from mammaliansystems are also commonly used, such as the mouse immunoglobulin heavychain enhancer.

Mammalian expression vector systems also typically include a selectablemarker gene. Examples of suitable markers include, the dihydrofolatereductase gene (DHFR), the thymidine kinase gene (TK), or prokaryoticgenes conferring drug resistance. The first two marker genes prefer theuse of mutant cell lines that lack the ability to grow without theaddition of thymidine to the growth medium. Transformed cells can thenbe identified by their ability to grow on non-supplemented media.Examples of prokaryotic drug resistance genes useful as markers includegenes conferring resistance to G418, mycophenolic acid and hygromycin.

The expression vectors containing the DNA segments of interest can betransferred into host cells by well-known methods, depending on the typeof cell production hosts. For example, calcium chloride transfection iscommonly utilized for prokaryotic host cells, whereas calcium phosphatetreatment, lipofection, or electroporation may be used for eukaryotichost cells. Other methods used to transform mammalian cell productionhosts include the use of polybrene, protoplast fusion, liposomes,electroporation, and microinjection (see, generally, Sambrook et al.,supra).

Once the expression vector has been introduced into an appropriate host,the host is maintained under conditions suitable for high levelexpression of the introduced mutant polynucleotide sequences to producethe mutant polypeptides. The expression vector is typically replicablein the host organisms either as episomes or as an integral part of thehost chromosomal DNA. Commonly, expression vectors will containselection markers, e.g., tetracycline or neomycin, to permit detectionof those cells transformed with the desired DNA sequences (see, e.g.,U.S. Pat. No. 4,704,362).

Therefore, in another aspect of the disclosure, mutant polynucleotidescan be generated by the process of reductive reassortment. The methodinvolves the generation of constructs containing consecutive sequences(original encoding sequences), their insertion into an appropriatevector, and their subsequent introduction into an appropriate host cell.The reassortment of the individual molecular identities occurs bycombinatorial processes between the consecutive sequences in theconstruct possessing regions of homology, or between quasi-repeatedunits. The reassortment process recombines and/or reduces the complexityand extent of the repeated sequences, and results in the production ofnovel molecular species. Various treatments may be applied to enhancethe rate of reassortment. These could include treatment withultra-violet light, or DNA damaging chemicals, and/or the use of hostcell lines displaying enhanced levels of “genetic instability”. Thus thereassortment process may involve homologous recombination or the naturalproperty of quasi-repeated sequences to direct their own evolution.

The cells are then propagated and “reductive reassortment” is effected.The rate of the reductive reassortment process may be stimulated by theintroduction of DNA damage if desired, in vivo reassortment is focusedon “inter-molecular” processes collectively referred to as“recombination” which in bacteria, is generally viewed as a“RecA-dependent” phenomenon. The disclosure can rely on recombinationprocesses of a host cell to recombine and re-assort sequences, or thecells' ability to mediate reductive processes to decrease the complexityof quasi-repeated sequences in the cell by deletion. This process of“reductive reassortment” occurs by an “intra-molecular”,RecA-independent process. The end result is a reassortment of themolecules into all possible combinations.

In one aspect, the host organism or cell includes a gram negativebacterium, a gram positive bacterium or a eukaryotic organism. Inanother aspect of the disclosure, the gram negative bacterium includesEscherichia coli, or Pseudomonas fluorescens. In another aspect of thedisclosure, the gram positive bacterium include Streptomyces diversa,Lactobacillus gasseri, Lactococcus lactis, Lactococcus cremoris, orBacillus subtilis. In another aspect of the disclosure, the eukaryoticorganism includes Saccharomyces cerevisiae, Schizosaccharomyces pombe,Pichia pastoris, Kluyveromyces lactis, Hansenula plymorpha, orAspergillus niger. As representative examples of appropriate hosts,there may be mentioned: bacterial cells, such as E. coli, Streptomyces,Salmonella typhimurium; fungal cells, such as yeast; insect cells suchas Drosophila S2 and Spodoptera Sf9; animal cells such as CHO, COS orBowes melanoma; adenoviruses; and plant cells. The selection of anappropriate host is deemed to be within the scope of those skilled inthe art from the teachings herein.

In addition to eukaryotic microorganisms such as yeast, mammalian tissuecell culture may also be used to express the mutant polypeptides of thepresent invention (see, Winnacker, “From Genes to Clones,” VCHPublishers, N.Y., N.Y. (1987)). Eukaryotic cells are preferred, becausea number of suitable host cell lines capable of secreting intactimmunoglobulins have been developed in the art, and include the CHO celllines, various COS cell lines, HeLa cells, myeloma cell lines, B-cellsor hybridomas. Expression vectors for these cells can include expressioncontrol sequences, such as an origin of replication, a promoter, anenhancer (Queen et al., Immunol. Rev., vol. 89, page 49, 1986), andnecessary processing information sites, such as ribosome binding sites,RNA splice sites, polyadenylation sites, and transcriptional terminatorsequences. Preferred expression control sequences are promoters derivedfrom immunoglobulin genes, cytomegalovirus, SV40, Adenovirus, BovinePapilloma Virus, and the like.

In one embodiment, the eukaryotic host cells are selected from CHO,HEK293, IM9, DS-1, THP-1, Hep G2, COS, NIH 3T3, C33a, A549, A375,SK-MEL-28, DU 145, PC-3, HCT 116, Mia PACA-2, ACHN, Jurkat, MM1, Ovcar3, HT 1080, Panc-1, U266, 769P, BT-474, Caco-2, HCC 1954, MDA-MB-468,LnCAP, NRK-49F, and SP2/0 cell lines; and mouse splenocytes and rabbitPBMC. In one aspect, the mammalian hoist cell is selected from a CHO orHEK293 cell line. In one specific aspect, the mammalian host cell is aCHO-S cell line. In another specific aspect, the mammalian system is aHEK293 cell line. In another embodiment, the eukaryotic host is a yeastcell system. In one aspect, the eukaryotic host is selected from S.cerevisiae yeast cells or picchia yeast cells.

In another embodiment, mammalian host cells may be created commerciallyby a contract research or custom manufacturing organization. Forexample, for recombinant antibodies or other proteins, Lonza (LonzaGroup Ltd, Basel, Switzerland) can create vectors to express theseproducts using the GS Gene Expression System™ technology with eitherCHOK1SV or NS0 cell production hosts. Host cells containing thepolynucleotides of interest can be cultured in conventional nutrientmedia modified as appropriate for activating promoters, selectingtransformants or amplifying genes. The culture conditions, such astemperature, pH and the like, are those previously used with the hostcell selected for expression, and will be apparent to the ordinarilyskilled artisan.

As discussed above, expression optimization for the conditionally activeASTR can be achieved by optimization of vectors used (vector components,such as promoters, splice sites, 5′ and 3′ termini and flankingsequences), gene modification of host cells to reduce gene deletions andrearrangements, evolution of host cell gene activities by in vivo or invitro methods of evolving relevant genes, optimization of hostglycosylating enzymes by evolution of relevant genes, and/or bychromosome wide host cell mutagenesis and selection strategies to selectfor cells with enhanced expression capabilities.

Protein expression can be induced by a variety of known methods, andmany genetic systems have been published for induction of proteinexpression. For example, with appropriate systems, the addition of aninducing agent will induce protein expression. Cells are then pelletedby centrifugation and the supernatant removed. Periplasmic protein canbe enriched by incubating the cells with DNAse, RNAse, and lysozyme.After centrifugation, the supernatant, containing the new protein, istransferred to a new multi-well tray and stored prior to assay.

Cells are typically harvested by centrifugation, disrupted by physicalor chemical means, and the resulting crude extract is retained forfurther purification. Microbial cells employed for expression ofproteins can be disrupted by any convenient method, includingfreeze-thaw cycling, sonication, mechanical disruption, or use of celllysing agents. Such methods are well known to those skilled in the art.The expressed polypeptide or fragment thereof can be recovered andpurified from recombinant cell cultures by methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Protein refolding steps can beused, as necessary, in completing configuration of the polypeptide. Ifdesired, high performance liquid chromatography (HPLC) can be employedfor final purification steps. The screening of a conditionally activeASTR can be aided by the availability of a convenient high throughputscreening or selection process. Cell surface display expression andscreening technology (for example, as defined above) can be employed toscreen mutant proteins for conditionally active ASTR.

Screening of Mutants to Identify Reversible or Nonreversible Mutants

Identifying desirable molecules is most directly accomplished bymeasuring protein activity at the permissive condition and the wild typecondition. The mutants with the largest ratio of activity(permissive/wild type) can then be selected and permutations of thepoint mutations are generated by combining the individual mutationsusing standard methods. The combined permutation protein library is thenscreened for those proteins displaying the largest differential activitybetween the permissive and wild type condition.

Activity of supernatants can be screened using a variety of methods, forexample using high throughput activity assays, such as fluorescenceassays, to identify protein mutants that are sensitive at whatevercharacteristic one desires (temperature, pH, etc). For example, toscreen for temporally sensitive mutants, the enzymatic or antibodyactivity of each individual mutant is determined at lower temperatures(such as 25 degrees Celsius), and at temperatures which the originalprotein functions (such as 37 degrees Celsius), using commerciallyavailable substrates. Screening can be carried out in a variety of mediasuch as serum and BSA, among others. Reactions can initially beperformed in a multi well assay format, such as a 96-well assay, andconfirmed using a different format, such as a 14 ml tube format.

In one aspect, the method further includes modifying at least one of thenucleic acids or polypeptides prior to testing the candidates forconditional biologic activity, in another aspect, the testing of step(c) further includes testing for improved expression of the polypeptidein a host cell or host organism, in a further aspect, the testing ofstep (c) further includes testing for enzyme activity within a pH rangefrom about pH 3 to about pH 12. In a further aspect, the testing of step(c) further includes testing for enzyme activity within a pH range fromabout pH 5 to about pH 10. In a further aspect, the testing of step (c)further includes testing for enzyme activity within a pH range fromabout pH 6 to about pH 8. In a further aspect, the testing of step (c)further includes testing for enzyme activity at pH 6.7 and pH 7.5. Inanother aspect, the testing of step (c) further includes testing forenzyme activity within a temperature range from about 4 degrees C. toabout 55 degrees C. In another aspect, the testing of step (c) furtherincludes testing for enzyme activity within a temperature range fromabout 15 degrees C. to about 47 degrees C. In another aspect, thetesting of step (c) further includes testing for enzyme activity withina temperature range from about 20 degrees C. to about 40 degrees C. Inanother aspect, the testing of step (c) further includes testing forenzyme activity at the temperatures of 25 degrees C. and 37 degrees C.In another aspect, the testing of step (c) further includes testing forenzyme activity under normal osmotic pressure, and aberrant (positive ornegative) osmotic pressure, In another aspect, the testing of step (c)further includes testing for enzyme activity under normal electrolyteconcentration, and aberrant (positive or negative) electrolyteconcentration. The electrolyte concentration to be tested is selectedfrom one of calcium, sodium, potassium, magnesium, chloride, bicarbonateand phosphate concentration, in another aspect, the testing of step (c)further includes testing for enzyme activity which results in astabilized reaction product.

In another aspect, the disclosure provides for a purified antibody thatspecifically binds to the polypeptide of the disclosure or a fragmentthereof, having enzyme activity. In one aspect, the disclosure providesfor a fragment of the antibody that specifically binds to a polypeptidehaving enzyme activity.

Antibodies and Antibody-Based Screening Methods

The disclosure provides isolated or recombinant antibodies thatspecifically bind to an enzyme of the disclosure. These antibodies canbe used to isolate, identify or quantify the enzymes of the disclosureor related polypeptides. These antibodies can be used to isolate otherpolypeptides within the scope the disclosure or other related enzymes.The antibodies can be designed to bind to an active site of an enzyme.Thus, the disclosure provides methods of inhibiting enzymes using theantibodies of the disclosure.

The antibodies can be used in immunoprecipitation, staining,immunoaffinity columns, and the like. If desired, nucleic acid sequencesencoding for specific antigens can be generated by immunization followedby isolation of polypeptide or nucleic acid, amplification or cloningand immobilization of polypeptide onto an array of the disclosure.Alternatively, the methods of the disclosure can be used to modify thestructure of an antibody produced by a cell to be modified, e.g., anantibody's affinity can be increased or decreased. Furthermore, theability to make or modify antibodies can be a phenotype engineered intoa cell by the methods of the disclosure.

Methods of immunization, producing and isolating antibodies (polyclonaland monoclonal) are known to those of skill in the art and described inthe scientific and patent literature, see, e.g., Coligan, CURRENTPROTOCOLS IN IMMUNOLOGY, Wiley/Greene, N Y (1991); Stites (eds.) BASICAND CLINICAL IMMUNOLOGY (7th ed.) Lange Medical Publications, Los Altos,Calif. (“Stites”); Goding, MONOCLONAL ANTIBODIES: PRINCIPLES ANDPRACTICE (2d ed.) Academic Press, New York, N.Y. (1986); Kohler (1975)“Continuous cultures of fused cells secreting antibody of predefinedspecificity”, Nature 256:495; Harlow (1988) ANTIBODIES, A LABORATORYMANUAL, Cold Spring Harbor Publications, New York. Antibodies also canbe generated in vitro, e.g., using recombinant antibody binding siteexpressing phage display libraries, in addition to the traditional invivo methods using animals. See, e.g., Hoogenboom (1997) “Designing andoptimizing library selection strategies for generating high-affinityantibodies”, Trends Biotechnol. 15:62-70; and Katz (1997) “Structuraland mechanistic determinants of affinity and specificity of ligandsdiscovered or engineered by phage display”, Annu. Rev. Biophys. Biomol.Struct. 26:27-45.

Polypeptides or peptides can be used to generate antibodies which bindspecifically to the polypeptides, e.g., the enzymes, of the disclosure.The resulting antibodies may be used in immunoaffinity chromatographyprocedures to isolate or purify the polypeptide or to determine whetherthe polypeptide is present in a biological sample. In such procedures, aprotein preparation, such as an extract, or a biological sample iscontacted with an antibody capable of specifically binding to one of thepolypeptides of the disclosure.

In immunoaffinity procedures, the antibody is attached to a solidsupport, such as a bead or other column matrix. The protein preparationis placed in contact with the antibody under conditions in which theantibody specifically binds to one of the polypeptides of thedisclosure. After a wash to remove non-specifically bound proteins, thespecifically bound polypeptides are eluted.

The ability of proteins in a biological sample to bind to the antibodymay be determined using any of a variety of procedures familiar to thoseskilled in the art. For example, binding may be determined by labelingthe antibody with a detectable label such as a fluorescent agent, anenzymatic label, or a radioisotope. Alternatively, binding of theantibody to the sample may be detected using a secondary antibody havingsuch a detectable label thereon. Particular assays include ELISA assays,sandwich assays, radioimmunoassays, and Western Blots.

Polyclonal antibodies generated against the polypeptides of thedisclosure can be obtained by direct injection of the polypeptides intoan animal or by administering the polypeptides to a non-human animal.The antibody so obtained will then bind the polypeptide itself. In thismanner, even a sequence encoding only a fragment of the polypeptide canbe used to generate antibodies which may bind to the whole nativepolypeptide. Such antibodies can then be used to isolate the polypeptidefrom cells expressing that polypeptide.

For preparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique, the trioma technique, thehuman B-cell hybridoma technique, and the EBV-hybridoma technique (see,e.g., Cole (1985) in Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, Inc., pp. 77-96).

Techniques described for the production of single chain antibodies (see,e.g., U.S. Pat. No. 4,946,778) can be adapted to produce single chainantibodies to the polypeptides of the disclosure. Alternatively,transgenic mice may be used to express humanized antibodies to thesepolypeptides or fragments thereof. Antibodies generated against thepolypeptides of the disclosure may be used in screening for similarpolypeptides (e.g., enzymes) from other organisms and samples. In suchtechniques, polypeptides from the organism are contacted with theantibody and those polypeptides which specifically bind the antibody aredetected. Any of the procedures described above may be used to detectantibody binding.

Screening Methodologies and “on-Line” Monitoring Devices

In practicing the methods of the disclosure, a variety of apparatus andmethodologies can be used to in conjunction with the polypeptides andnucleic acids of the disclosure, e.g., to screen polypeptides for enzymeactivity, to screen compounds as potential modulators, e.g., activatorsor inhibitors, of an enzyme activity, for antibodies that bind to apolypeptide of the disclosure, for nucleic acids that hybridize to anucleic acid of the disclosure, to screen for cells expressing apolypeptide of the disclosure and the like.

Arrays, or “Biochips”

Nucleic acids or polypeptides of the disclosure can be immobilized to orapplied to an array. Arrays can be used to screen for or monitorlibraries of compositions (e.g., small molecules, antibodies, nucleicacids, etc.) for their ability to bind to or modulate the activity of anucleic acid or a polypeptide of the disclosure. For example, in oneaspect of the disclosure, a monitored parameter is transcript expressionof an enzyme gene. One or more, or, all the transcripts of a cell can bemeasured by hybridization of a sample including transcripts of the cell,or, nucleic acids representative of or complementary to transcripts of acell, by hybridization to immobilized nucleic acids on an array, or“biochip.” By using an “array” of nucleic acids on a microchip, some orall of the transcripts of a cell can be simultaneously quantified.Alternatively, arrays including genomic nucleic acid can also be used todetermine the genotype of a newly engineered strain made by the methodsof the disclosure. Polypeptide arrays” can also be used tosimultaneously quantify a plurality of proteins. The present disclosurecan be practiced with any known “array,” also referred to as a“microarray” or “nucleic acid array” or “polypeptide array” or “antibodyarray” or “biochip,” or variation thereof. Arrays are generically aplurality of “spots” or “target elements,” each target element includinga defined amount of one or more biological molecules, e.g.,oligonucleotides, immobilized onto a defined area of a substrate surfacefor specific binding to a sample molecule, e.g., mRNA transcripts.

In practicing the methods of the disclosure, any known array and/ormethod of making and using arrays can be incorporated in whole or inpart, or variations thereof, as described, for example, in U.S. Pat.Nos. 6,277,628; 6,277,489; 6,261,776; 6,258,606; 6,054,270; 6,048,695;6,045,996; 6,022,963; 6,013,440; 5,965,452; 5,959,098; 5,856,174;5,830,645; 5,770,456; 5,632,957; 5,556,752; 5,143,854; 5,807,522;5,800,992; 5,744,305; 5,700,637; 5,556,752; 5,434,049; see also, e.g.,WO 99/51773; WO 99/09217; WO 97/46313; WO 96/17958; see also, e.g.,Johnston (1998) “Gene chips: Array of hope for understanding generegulation”, Curr. Biol. 8:R171-R174; Schummer (1997) “InexpensiveHandheld Device for the Construction of High-Density Nucleic AcidArrays”, Biotechniques 23:1087-1092; Kern (1997) “Direct hybridizationof large-insert genomic clones on high-density gridded cDNA filterarrays”, Biotechniques 23:120-124; Solinas-Toldo (1997) “Matrix-BasedComparative Genomic Hybridization: Biochips to Screen for GenomicImbalances”, Genes, Chromosomes & Cancer 20:399-407; Bowtell (1999)“Options Available-From Start to Finish—for Obtaining Expression Data byMicroarray”, Nature Genetics Supp. 21:25-32. See also published U.S.patent applications Nos. 20010018642; 20010019827; 20010016322;20010014449; 20010014448; 20010012537; 20010008765.

Capillary Arrays

Capillary arrays, such as the GIGAMATRIX™ Diversa Corporation, SanDiego, Calif., can be used in the methods of the disclosure. Nucleicacids or polypeptides of the disclosure can be immobilized to or appliedto an array, including capillary arrays. Arrays can be used to screenfor or monitor libraries of compositions (e.g., small molecules,antibodies, nucleic acids, etc.) for their ability to bind to ormodulate the activity of a nucleic acid or a polypeptide of thedisclosure. Capillary arrays provide another system for holding andscreening samples. For example, a sample screening apparatus can includea plurality of capillaries formed into an array of adjacent capillaries,wherein each capillary includes at least one wall defining a lumen forretaining a sample. The apparatus can further include interstitialmaterial disposed between adjacent capillaries in the array, and one ormore reference indicia formed within of the interstitial material. Acapillary for screening a sample, wherein the capillary is adapted forbeing bound in an array of capillaries, can include a first walldefining a lumen for retaining the sample, and a second wall formed of afiltering material, for filtering excitation energy provided to thelumen to excite the sample. A polypeptide or nucleic acid, e.g., aligand, can be introduced into a first component into at least a portionof a capillary of a capillary array. Each capillary of the capillaryarray can include at least one wall defining a lumen for retaining thefirst component. An air bubble can be introduced into the capillarybehind the first component. A second component can be introduced intothe capillary, wherein the second component is separated from the firstcomponent by the air bubble. A sample of interest can be introduced as afirst liquid labeled with a detectable particle into a capillary of acapillary array, wherein each capillary of the capillary array includesat least one wall defining a lumen for retaining the first liquid andthe detectable particle, and wherein the at least one wall is coatedwith a binding material for binding the detectable particle to the atleast one wall. The method can further include removing the first liquidfrom the capillary tube, wherein the bound detectable particle ismaintained within the capillary, and introducing a second liquid intothe capillary tube. The capillary array can include a plurality ofindividual capillaries including at least one outer wall defining alumen. The outer wall of the capillary can be one or more walls fusedtogether. Similarly, the wall can define a lumen that is cylindrical,square, hexagonal or any other geometric shape so long as the walls forma lumen for retention of a liquid or sample. The capillaries of thecapillary array can be held together in close proximity to form a planarstructure. The capillaries can be bound together, by being fused (e.g.,where the capillaries are made of glass), glued, bonded, or clampedside-by-side. The capillary array can be formed of any number ofindividual capillaries, for example, a range from 100 to 4,000,000capillaries. A capillary array can form a micro titer plate having about100,000 or more individual capillaries bound together.

Engineering Conditionally Active Antibodies

Conditionally active antibodies may be engineered to generatemultispecific conditionally active antibodies. The multispecificantibody may be an antibody with polyepitopic specificity, as describedin WO 2013/170168. Multispecific antibodies include, but are not limitedto, an antibody including a heavy chain variable domain (V_(H)) and alight chain variable domain (V_(L)), where the V_(H)V_(L) unit haspolyepitopic specificity, antibodies having two or more V_(L) and V_(H)domains where each V_(H)V_(L) unit binds to a different epitope,antibodies having two or more single variable domains with each singlevariable domain binding to a different epitope, and antibodies includingone or more antibody fragments as well as antibodies including antibodyfragments that have been linked covalently or non-covalently.

To construct multispecific antibodies, including bispecific antibodies,antibody fragments having at least one free sulfhydryl group areobtained. The antibody fragments may be obtained from full-lengthconditionally active antibodies. The conditionally active antibodies maybe digested enzymatically to produce antibody fragments. Exemplaryenzymatic digestion methods include, but are not limited to, pepsin,papain and Lys-C. Exemplary antibody fragments include, but are notlimited to, Fab, Fab′, F(ab′)2, Fv, diabodies (Db); tandem diabodies(taDb), linear antibodies (see U.S. Pat. No. 5,641,870, Example 2;Zapata et al., Protein Eng., vol. 8, pages 1057-1062 (1995)); one-armedantibodies, single variable domain antibodies, minibodies (Olafsen et al(2004) Protein Eng. Design & Sel., vol. 17, pages 315-323), single-chainantibody molecules, fragments produced by a Fab expression library,anti-idiotypic (anti-Id) antibodies, complementary determining regions(CDRs), and epitope-binding fragments. Antibody fragments may also beproduced using DNA recombinant technology. The DNA encoding the antibodyfragments may be cloned into plasmid expression vectors or phagemidvectors and expressed directly in E. Coli. Antibody enzymatic digestionmethods, DNA cloning and recombinant protein expression methods are wellknown to those skilled in the art.

Antibody fragments may be purified using conventional techniques and maybe subjected to reduction to generate a free thiol group. Antibodyfragments having a free thiol group may be reacted with a cross-linker,for example, bis-maleimide. Such crosslinked antibody fragments arepurified and then reacted with a second antibody fragment having a freethiol group. The final product in which two antibody fragments arecrosslinked is purified. In certain embodiments, each antibody fragmentis a Fab and the final product, in which the two Fabs are linked throughbis-maleimide, is referred to herein as bismaleimido-(thio-Fab)2, orbis-Fab. Such multispecific antibodies and antibody analogs, includingbis-Fabs, can be exploited to quickly synthesize a large number ofantibody fragment combinations, or structural variants of nativeantibodies or particular antibody/fragment combinations.

Multispecific antibodies can be synthesized with modified cross-linkerssuch that additional functional moieties may be attached to themultispecific antibodies. Modified cross-linkers allow for attachment ofany sulfhydryl-reactive moiety. In one embodiment,N-succinimidyl-S-acetylthioacetate (SATA) is attached to bis-maleimideto form bis-maleimido-acetylthioacetate (BMata). After deprotection ofthe masked thiol group, any functional group having asulfhydryl-reactive (or thiol-reactive) moiety may be attached to themultispecific antibodies.

Exemplary thiol-reactive reagents include a multifunctional linkerreagent, a capture, i.e. an affinity, label reagent (e.g. abiotin-linker reagent), a detection label (e.g. a fluorophore reagent),a solid phase immobilization reagent (e.g. SEPHAROSE™, polystyrene, orglass), or a drug-linker intermediate. One example of a thiol-reactivereagent is N-ethyl maleimide (NEM). Such multispecific antibodies orantibody analogs having modified cross-linkers may be further reactedwith a drug moiety reagent or other label. Reaction of a multispecificantibody or antibody analog with a drug-linker intermediate provides amultispecific antibody-drug conjugate or antibody analog-drug conjugate,respectively.

Other techniques for making multispecific antibodies may also be used inthe present invention. References describing these techniques include:(1) Milstein and Cuello, Nature, vol. 305, page 537 (1983)), WO93/08829, and Traunecker et al., EMBO J., vol. 10, page 3655 (1991) onrecombinant co-expression of two immunoglobulin heavy chain-light chainpairs having different specificities; (2) U.S. Pat. No. 5,731,168 on“knob-in-hole” engineering; (3) WO 2009/089004A1 on engineeringelectrostatic steering effects for making antibody Fc-heterodimericmolecules; (4) U.S. Pat. No. 4,676,980, and Brennan et al., Science,vol. 229, page 81 (1985) on cross-linking two or more antibodies orfragments; (5) Kostelny et al., J. Immunol., vol. 148, pages 1547-1553(1992) on using leucine zippers to produce bi-specific antibodies; (6)Hollinger et al., Proc. Natl. Acad. Sci. USA, vol. 90, pages 6444-6448(1993) on using “diabody” technology for making bispecific antibodyfragments; (7) Gruber et al., J. Immunol., vol. 152, page 5368 (1994) onusing single-chain Fv (sFv) dimers; (8) Tutt et al. J. Immunol. 147: 60(1991) on preparing trispecific antibodies; and (9) US 2006/0025576A1and Wu et al. Nature Biotechnology, vol. 25, pages 1290-1297 (2007) onengineered antibodies with three or more functional antigen bindingsites, including “Octopus antibodies” or “dual-variable domainimmunoglobulins” (DVDs).

Multispecific antibodies of the present invention may also be generatedas described in WO/2011/109726.

In one embodiment, a conditionally active antibody for crossing theblood-brain barrier (BBB) is engineered to make a multispecific antibody(e.g. a bispecific antibody). This multispecific antibody includes afirst antigen binding site which binds a BBB-R and a second antigenbinding site which binds a brain antigen. At least the first antigenbinding site for BBB-R is conditionally active. A brain antigen is anantigen expressed in the brain, which can be targeted with an antibodyor small molecule. Examples of such antigens include, withoutlimitation: beta-secretase 1 (BACE1), amyloid beta (Abeta), epidermalgrowth factor receptor (EGFR), human epidermal growth factor receptor 2(HER2), Tau, apolipoprotein E4 (ApoE4), alpha-synuclein, CD20,huntingtin, prion protein (PrP), leucine rich repeat kinase 2 (LRRK2),parkin, presenilin 1, presenilin 2, gamma secretase, death receptor 6(DR6), amyloid precursor protein (APP), p′75 neurotrophin receptor(p75NTR), and caspase 6. In one embodiment, the antigen is BACE1.

The BBB has endogenous transport systems that are mediated by a BBBreceptor (BBB-R), which is a specific receptor that allows transport ofmacromolecules across the BBB. For example, an antibody that can bind toa BBB-R may be transported across BBB using the endogenous transportsystems. Such an antibody may serve as a vehicle for transport of drugsor other agents across BBB by using the endogenous BBB receptor mediatedtransport system that traverses the BBB. Such antibodies need not havehigh affinity to a BBB-R. Antibodies that are not conditionally activeantibodies with low affinities for BBB-R have been described as crossingthe BBB more efficiently than a high affinity antibody, as described inUS 2012/0171120.

Another method for engineering antibodies to enter the brain is toengineer antibodies to be delivered to the brain via the central nervoussystem lymphatic vessels. Thus, the antibodies can be engineered to bindto or mimic immune cells such as T-cells, or synovial or cerebrospinalfluids that travel to the central nervous system via lymphatic vessels.Details of the lymphatic vessels of the central nervous system aredescribed in, for example, Louveau, A., et al., “Structural andfunctional features of central nervous system lymphatic vessels,” Nature523, pp. 337-341, 16 Jul. 2015 and the articles citing this article thatare publicly available as of the date of filing of this application.

Unlike traditional antibodies, conditionally active antibodies are notrequired to have low affinity for BBB-R to cross the BBB and remaininside the brain. Conditionally active antibodies can have high affinityfor the BBB-R on the blood side of the BBB, and little or no affinity onthe brain side of the BBB. Drugs, such as drug conjugates, may becoupled to a conditionally active antibody to be transported with theantibody across the BBB into the brain.

A BBB-R is a transmembrane receptor protein expressed on brainendothelial cells which is capable of transporting molecules across theblood-brain barrier. Examples of BBB-R include transferrin receptor(TfR), insulin receptor, insulin-like growth factor receptor (IGF-R),low density lipoprotein receptors including without limitation lowdensity lipoprotein receptor-related protein 1 (LRP1) and low densitylipoprotein receptor-related protein 8 (LRP8), and heparin-bindingepidermal growth factor-like growth factor (HB-EGF). An exemplary BBB-Rherein is a transferrin receptor (TfR). The TfR is a transmembraneglycoprotein (with a molecular weight of about 180,000) composed of twodisulphide-bonded sub-units (each of apparent molecular weight of about90,000) involved in iron uptake in vertebrates.

In some embodiments, the present invention provides a conditionallyactive antibody generated from a parent or wild-type antibody against aBBB-R. The conditionally active antibody binds the BBB-R on the bloodside of the BBB, and has a lower affinity to the BBB-R than the parentor wild-type antibody on the brain side of the BBB. In some otherembodiments, the conditionally active antibody has affinity to the BBB-Rthan the wild type or parent antibody on the blood side of the BBB, andhas no affinity to the BBB-R on the brain side of the BBB.

Blood plasma is a body fluid that is very different from brainextracellular fluid (ECF). As discussed by Somjen (“Ions in the Brain:Normal Function, Seizures, and Stroke,” Oxford University Press, 2004,pages 16 and 33) and Redzic (“Molecular biology of the blood-brain andthe blood-cerebrospinal fluid barriers: similarities and differences,”Fluids and Barriers of the CNS, vol. 8:3, 2011), the brain extracellularfluid has significantly less K⁺, more Mg²⁺ and H⁺ than blood plasma. Thedifferences in ion concentrations between blood plasma and brain ECFlead to significant differences in osmotic pressure and osmolalitybetween the two fluids. Table 1 shows the concentrations of common ionsin millimoles for both blood plasma and brain ECF.

TABLE 1 Common ions in plasma (arterial plasma) and brain extracellularfluid (CSF) ARTERIAL PLASMA CSF HUMAN RAT HUMAN RAT Na⁺ 150 148 147 152K⁺ 4.6 5.3 2.9 3.4 Ca, total 2.4 3.1 1.14 1.1 Ca²⁺, free 1.4 1.5 1.0 1.0pCa Mg, total 0.86 0.8 1.15 1.3 Mg²⁺, free 0.47 0.44 0.7 0.88 H⁺0.000039 0.000032 0.000047 0.00005 pH 7.41 7.5 7.3 7.3 Cl⁻ 99 119 HCO₃ ⁻26.8 31 23.3 28

Brain ECF also contains significantly more lactate than blood plasma andsignificantly less glucose than blood plasma (Abi-Saab et al., “StrikingDifferences in Glucose and Lactate Levels Between Brain ExtracellularFluid and Plasma in Conscious Human Subjects: Effects of Hyperglycemiaand Hypoglycemia,” Journal of Cerebral Blood Flow & Metabolism, vol. 22,pages 271-279, 2002).

Thus, there are several physiological conditions that are differentbetween the two sides of the BBB, such as pH, concentrations of varioussubstances (such as lactose, glucose, K+, Mg2+), osmotic pressure andosmolality. For the physiological condition of pH, human blood plasmahas a higher pH than human brain ECF. For the physiological condition ofK+ concentration, brain ECF has a lower K+ concentration than humanblood plasma. For the physiological condition of Mg2+ concentration, thehuman brain ECF has significantly more Mg2+ than human blood plasma. Forthe physiological condition of osmotic pressure, the human brain ECF hasan osmotic pressure that is different from that of human blood plasma.In some embodiments, the physiological conditions of brain ECF may bethe composition, pH, osmotic pressure and osmolality of brain ECF ofpatients with a particular neurological disorder, which may be differentfrom the physiological condition of the brain ECF of the generalpopulation.

The present invention thus provides a method for evolving a DNA thatencodes a template antibody against a BBB-R to create a mutant DNAlibrary. The mutant DNA library is then expressed to obtain mutantantibodies. The mutant antibodies are screened for a conditionallyactive antibody that has binds to the BBB-R under at least one bloodplasma physiological condition and has a low or no affinity to the BBB-Runder at least one brain physiological condition in the brain ECFcompared to the template antibody. Thus, the selected mutant antibodyhas a low or high affinity to the BBB-R at the blood plasma side and alow or no affinity to the BBB-R at the brain ECF side. This selectedmutant antibody is useful as a conditionally active antibody fortransport across the BBB.

Such a conditionally active antibody is advantageous for crossing theBBB and remaining in the brain ECF. The low affinity to the BBB-R at thebrain side lowers the rate (or removes) the conditionally activeantibody is transported back across the BBB out of the brain and backinto the blood relative to the template antibody.

In some other embodiments, the present invention provides a method forevolving a DNA that encodes a template antibody against a BBB-R tocreate a mutant DNA library. The mutant DNA library is then expressed toobtain mutant antibodies. The mutant antibodies are screened for aconditionally active antibody that binds to the BBB-R under at least oneblood plasma physiological condition and little or no affinity to theBBB-R under at least one brain physiological condition. Thus, theselected mutant antibody has affinity to the BBB-R at the plasma sideand little or no affinity to the BBB-R at the brain ECF side. Thisselected mutant antibody is a conditionally active antibody.

Such a conditionally active antibody is advantageous in crossing the BBBand remaining in the brain ECF. After binding to the BBB-R at the bloodplasma side, the conditionally active antibody is transported across theBBB, and the little to no affinity to the BBB-R at the brain ECF sidemeans that the conditionally active antibody is unlikely to betransported out of the brain.

The affinity of the conditionally active antibody to a BBB-R may bemeasured by its half maximal inhibitory concentration (IC50), which is ameasure of how much of the antibody is needed to inhibit the binding ofa known BBB-R ligand to the BBB-R by 50%. A common approach is toperform a competitive binding assay, such as competitive ELISA assay. Anexemplary competitive ELISA assay to measure IC50 on TfR (a BBB-R) isone in which increasing concentrations of anti-TfR antibody competeagainst biotinylated TfR^(A) for binding to TfR. The anti-TfR antibodycompetitive ELISA may be performed in Maxisorp plates (Neptune, N.J.)coated with 2.5 μg/ml of purified murine TfR extracellular domain in PBSat 4° C. overnight. Plates are washed with PBS/0.05% Tween 20 andblocked using Superblock blocking buffer in PBS (Thermo Scientific,Hudson, N.H.). A titration of each individual anti-TfR antibody (1:3serial dilution) is combined with biotinylated anti-TfR^(A) (0.5 nMfinal concentration) and added to the plate for 1 hour at roomtemperature. Plates are washed with PBS/0.05% Tween 20, andHRP-streptavidin (Southern Biotech, Birmingham) is added to the plateand incubated for 1 hour at room temperature. Plates are washed withPBS/0.05% Tween 20, and biotinylated anti-TfR^(A) bound to the plate isdetected using TMB substrate (BioFX Laboratories, Owings Mills).

A high IC50 indicates that more of the conditionally active antibody isrequired to inhibit binding of the known ligand of a BBB-R, and thusthat the antibody's affinity for that BBB-R is relatively low.Conversely, a low IC50 indicates that less of the conditionally activeantibody is required to inhibit binding of the known ligand, and thusthat the antibody's affinity for that BBB-R is relatively high.

In some embodiments, the IC50 of the conditionally active antibodiesfrom a BBB-R in the blood plasma may be from about 1 nM to about 100 μM,or from about 5 nM to about 100 μM, or from about 50 nM to about 100 μM,or from about 100 nM to about 100 μM, or from about 5 nM to about 10 μM,or from about 30 nM to about 1 μM, or from about 50 nM to about 1 μM.

Conditionally Active Biologic Proteins for Synovial Fluid

Joint diseases are a major cause of disability and early retirement inthe industrialized countries. Joint diseases often lead to damage at ajoint which is difficult to repair. Synovial fluid is a body fluid thatis found in the synovial cavity of the joints (e.g., knee, hip,shoulder) of a human or animal body between the cartilage and synoviumof facing articulating surfaces. Synovial fluid provides nourishment tothe cartilage and also serves as a lubricant for the joints. The cellsof the cartilage and synovium secrete fluid that serve as a lubricantbetween the articulating surfaces. Human synovial fluid includesapproximately 85% water. It is derived from the dialysate of bloodplasma, which itself is made up of water, dissolved proteins, glucose,clotting factors, mineral ions, hormones, etc. Proteins such as albuminand globulins are present in synovial fluid and are believed to play animportant role in the lubricating the joint area. Some other proteinsare also found in human synovial fluid, including the glycoproteins suchas alpha-1-acid glycoprotein (AGP), alpha-1-antitrypsin (A1AT) andlubricin.

Synovial fluid has a composition that is very different from other partsof the body. Thus, synovial fluid has physiological conditions that aredifferent from other parts of the body, such as the blood plasma. Forexample, synovial fluid has less than about 10 mg/dL of glucose whereasthe mean normal glucose level in human blood plasma is about 100 mg/dL,fluctuating within a range between 70 and 100 mg/dL throughout the day.In addition, the total protein level in the synovial fluid is about onethird of the blood plasma protein level since large molecules such asproteins do not easily pass through the synovial membrane into thesynovial fluid. It has also been found that the pH of human synovialfluid is higher than the pH in human plasma (Jebens et al., “On theviscosity and pH of synovial fluid and the pH of blood,” The Journal ofBone and Joint Surgery, vol. 41 B, pages 388-400, 1959; Farr et al.,“Significance of the hydrogen ion concentration in synovial fluid inRheumatoid Arthritis,” Clinical and Experimental Rheumatology, vol. 3,pages 99-104, 1985).

Thus, the synovial fluid has several physiological conditions that aredifferent from those of the other parts of body, such as thephysiological conditions in the blood plasma. The synovial fluid has apH that is higher than other parts of the body, especially the bloodplasma. The synovial fluid has a lower concentration of glucose thanother parts of the body, such as blood plasma. The synovial fluid alsohas a lower concentration of protein than other parts of the body, suchas blood plasma.

Several antibodies have been used to treat joint disease by introducingthe antibodies into the synovial fluid. For example, the synovial fluidin an injured joint is known to contain many factors which have aninfluence on the progression of osteoarthritis (see, for example,Fernandes, et al., “The Role of Cytokines in OsteoarthritisPathophysiology”, Biorheology, vol. 39, pages 237-246, 2002). Cytokines,such as Interleukin-1 (IL-I) and Tumor Necrosis Factor-α (TNF-α), whichare produced by activated synoviocytes, are known to upregulate matrixmetalloproteinase (MMP) gene expression. Upregulation of MMP leads todegredation of the matrix and non-matrix proteins in the joints.Antibodies that neutralize cytokines may stop the progression ofosteoarthritis.

Using antibodies as drug is a promising strategy for the treatment ofjoint diseases. For example, antibodies (such as antibody againstaggrecan or aggrecanase) have been developed to treat osteoarthritis,which has by far the greatest prevalence among joint diseases(WO1993/022429A1). An antibody against acetylated high-mobility groupbox 1 (HMGB1) has been developed for diagnosis or treatment of jointdiseases that are inflammatory, autoimmune, neurodegenerative ormalignant diseases/disorders, such as arthritis. This antibody may beused to detect the acetylated form of HMGB1 in synovial fluid (WO2011/157905A1). Another antibody (CD20 antibody) has also been developedto treat damage to connective tissue and cartilage of the joints.

However, the antigens of these antibodies are often expressed in otherparts of the body carrying important physiological functions. Antibodiesagainst these antigens, though efficacious in treating joint diseases,may also significantly interfere with the normal physiological functionsof these antigens in other parts of the body. Therefore, severe sideeffects may be experienced by patients. It is thus desirable to developtherapeutics, such as antibodies against cytokines or other antigensthat can preferentially bind to their antigens (proteins or othermacromolecules) at higher affinity in the synovial fluid, while notbinding or only weakly binding to the same antigens in other parts ofthe body in order to reduce side effects.

Such conditionally active biologic proteins may be conditionally activeantibodies. In some embodiments, the present invention also providesconditionally active biologic proteins that are proteins other thanantibodies. For example, a conditionally active immune regulator may bedeveloped by the present invention for preferentially regulating theimmune response in the synovial fluid, which may less or no effect onthe immune response at other parts of the body.

The conditionally active biologic proteins may be conditionally activesuppressors of cytokine signaling (SOCS). Many of these SOCS areinvolved in inhibiting the JAK-STAT signaling pathway. The conditionallyactive suppressors of cytokine signaling can preferentially suppress thecytokine signaling in the synovial fluid, while not or to a lesserextent suppressing the cytokine signaling in other parts of the body.

In some embodiments, the present invention provides a conditionallyactive biologic protein derived from a wild-type biologic protein. Theconditionally active biologic protein has a lower activity under atleast one physiological condition in certain parts of the body such asin blood plasma than the wild-type biologic protein, and has a higheractivity than the wild-type biologic protein under at least onephysiological condition in the synovial fluid. Such conditionally activebiologic proteins can preferentially function in the synovial fluid, butnot or to a lesser extent act upon other parts of the body.Consequently, such conditionally active biologic proteins may havereduced side effects.

In some embodiments, the conditionally active biologic proteins areantibodies against an antigen in or exposed to synovial fluid. Suchantigens may be any proteins involved in immune response/inflammation ina joint disease, though the antigen is often a cytokine. Theconditionally active antibody has a lower affinity to the antigen thanthe wild-type antibody for the same antigen under at least onephysiological condition in other parts of the body (such as bloodplasma), while has higher affinity for the antigen than the wild-typeantibody under at least one physiological condition of synovial fluid.Such conditionally active antibodies can bind weakly or not at all tothe antigen in other parts of the body, but bind, for example bindstrongly and tightly or bind stronger to the antigen in synovial fluid.

Conditionally Active Biologic Proteins for Tumors

Cancer cells in a solid tumor are able to form a tumor microenvironmentin their surroundings to support the growth and metastasis of the cancercells. A tumor microenvironment is the cellular environment in which thetumor exists, including surrounding blood vessels, immune cells,fibroblasts, other cells, soluble factors, signaling molecules, anextracellular matrix, and mechanical cues that can promote neoplastictransformation, support tumor growth and invasion, protect the tumorfrom host immunity, foster therapeutic resistance, and provide nichesfor dormant metastases to thrive. The tumor and its surroundingmicroenvironment are closely related and interact constantly. Tumors caninfluence their microenvironment by releasing extracellular signals,promoting tumor angiogenesis and inducing peripheral immune tolerance,while the immune cells in the microenvironment can affect the growth andevolution of cancerous cells. See Swarts et al. “Tumor MicroenvironmentComplexity: Emerging Roles in Cancer Therapy,” Cancer Res, vol., 72,pages 2473-2480, 2012.

The tumor microenvironment is often hypoxic. As the tumor massincreases, the interior of the tumor grows farther away from existingblood supply, which leads to difficulties in fully supplying oxygen tothe tumor microenvironment. The partial oxygen pressure in the tumorenvironment is below 5 mm Hg in more than 50% of locally advanced solidtumors, in comparison with a partial oxygen pressure at about 40 mm Hgin blood plasma. In contrast, other parts of the body are not hypoxic.The hypoxic environment leads to genetic instability, which isassociated with cancer progression, via downregulating nucleotideexcision repair and mismatch repair pathways. Hypoxia also causes theupregulation of hypoxia-inducible factor 1 alpha (HIF1-α), which inducesangiogenesis, and is associated with poorer prognosis and the activationof genes associated with metastasis. See Weber et al., “The tumormicroenvironment,” Surgical Oncology, vol. 21, pages 172-177, 2012 andBlagosklonny, “Antiangiogenic therapy and tumor progression,” CancerCell, vol. 5, pages 13-17, 2004.

In addition, tumor cells tend to rely on energy generated from lacticacid fermentation, which does not require oxygen. So tumor cells areless likely to use normal aerobic respiration that does require oxygen.A consequence of using lactic acid fermentation is that the tumormicroenvironment is acidic (pH 6.5-6.9), in contrast to other parts ofthe body which are typically either neutral or slightly basic. Forexample, human blood plasma has a pH of about 7.4. See Estrella et al.,“Acidity Generated by the Tumor Microenvironment Drives Local Invasion,”Cancer Research, vol. 73, pages 1524-1535, 2013. The nutrientavailability in the tumor microenvironment is also low due to therelatively high nutrient demand of the proliferating cancer cells, incomparison with cells located in other parts of the body.

Further, the tumor microenvironment also contains many distinct celltypes not commonly found in other parts of the body. These cell typesinclude endothelial cells and their precursors, pericytes, smooth musclecells, Wbroblasts, carcinoma-associated Wbroblasts, myoWbroblasts,neutrophils, eosinophils, basophils, mast cells, T and B lymphocytes,natural killer cells and antigen presenting cells (APC) such asmacrophages and dendritic cells (Lorusso et al., “The tumormicroenvironment and its contribution to tumor evolution towardmetastasis,” Histochem Cell Biol, vol. 130, pages 1091-1103, 2008).

Accordingly, the tumor microenvironment has at least severalphysiological conditions that are different from those of other parts ofbody, such as the physiological conditions in blood plasma. The tumormicroenvironment has a pH (acidic) that is lower than other parts of thebody, especially the blood plasma (pH 7.4). The tumor microenvironmenthas a lower concentration of oxygen than other parts of the body, suchas blood plasma. Also, the tumor microenvironment has a lower nutrientavailability than other parts of the body, especially the blood plasma.The tumor microenvironment also has some distinct cell types that arenot commonly found in other parts of the body, especially the bloodplasma.

Some cancer drugs include antibodies that can penetrate into the tumormicroenvironment and act upon the cancer cells therein. Antibody-basedtherapy for cancer is well established and has become one of the mostsuccessful and important strategies for treating patients withhaematological malignancies and solid tumors. There is a broad array ofcell surface antigens that are expressed by human cancer cells that areoverexpressed, mutated or selectively expressed in cancer cells comparedwith normal tissues. These cell surface antigens are excellent targetsfor antibody cancer therapy.

Cancer cell surface antigens that may be targeted by antibodies fallinto several different categories. Haematopoietic differentiationantigens are glycoproteins that are usually associated with clusters ofdifferentiation (CD) groupings and include CD20, CD30, CD33 and CD52.Cell surface differentiation antigens are a diverse group ofglycoproteins and carbohydrates that are found on the surface of bothnormal and tumor cells. Antigens that are involved in growth anddifferentiation signaling are often growth factors and growth factorreceptors. Growth factors that are targets for antibodies in cancerpatients include CEA2, epidermal growth factor receptor (EGFR; alsoknown as ERBB1)12, ERBB2 (also known as HER2)13, ERBB3 (REF. 18), MET(also known as HGFR)19, insulin-like growth factor 1 receptor (IGF1R)20,ephrin receptor A3 (EPHA3)21, tumor necrosis factor (TNF)-relatedapoptosis-inducing ligand receptor 1 (TRAILR1; also known as TNFRSF10A),TRAILR2 (also known as TNFRSF10B) and receptor activator of nuclearfactor-κB ligand (RANKL; also known as TNFSF11)22. Antigens involved inangiogenesis are usually proteins or growth factors that support theformation of new microvasculature, including vascular endothelial growthfactor (VEGF), VEGF receptor (VEGFR), integrin αVβ3 and integrin α5β1(REF. 10). Tumor stroma and the extracellular matrix are indispensablesupport structures for a tumor. Stromal and extracellular matrixantigens that are therapeutic targets include fibroblast activationprotein (FAP) and tenascin. See Scott et al., “Antibody therapy ofcancer,” Nature Reviews Cancer, vol. 12, pages 278-287, 2012.

In addition to antibodies, other biologic proteins have also shownpromise in treating cancers. Examples include tumor suppressors such asRetinoblastoma protein (pRb), p53, pVHL, APC, CD95, ST5, YPEL3, ST7, andST14. Some proteins that induce apoptosis in cancer cells may also beintroduced into tumors for shrinking the size of tumors. There are atleast two mechanisms that can induce apoptosis in tumors: the tumornecrosis factor-induced mechanism and the Fas-Fas ligand-mediatedmechanism. At least some of the proteins involved in either of the twoapoptotic mechanisms may be introduced to tumors for treatment.

Cancer stem cells are cancer cells that have the ability to give rise toall cell types found in a particular cancer sample, and are thereforetumor-forming. They may generate tumors through the stem cell processesof self-renewal and differentiation into multiple cell types. It isbelieved that cancer stem cells persist in tumors as a distinctpopulation and cause relapse and metastasis by giving rise to newtumors. Development of specific therapies targeted at cancer stem cellsmay improve the survival and quality of life of cancer patients,especially for sufferers of metastatic disease.

These drugs for treating tumors often interfere with normalphysiological functions in other parts of the body besides tumors. Forexample, proteins inducing apoptosis in tumors may also induce apoptosisin some other parts of the body thus causing side effects. Inembodiments where an antibody is used to treat tumors, the antigen ofthe antibody may also be expressed in other parts of the body where theyperform normal physiological functions. For example, monoclonal antibodybevacizumab (targeting vascular endothelial growth factor) to stop tumorblood vessel growth. This antibody can also prevent blood vessel growthor repair in other parts of the body, thus causing bleeding, poor woundhealing, blood clots, and kidney damage. Development of a conditionallyactive biologic protein that concentrates on targeting mainly or solelytumors is highly desirable for more effective tumor therapies.

In some embodiments, the present invention provides a conditionallyactive biologic protein generated from a wild-type biologic protein thatmay be a candidate for tumor treatment. The conditionally activebiologic protein has lower activity under at least one physiologicalcondition in parts of the body other than the tumor microenvironmentsuch as blood plasma than the wild-type biologic protein, while it hashigher activity under at least one physiological condition in the tumormicroenvironment than the wild-type biologic protein. Such conditionallyactive biologic proteins can preferentially act upon cancer cells in thetumor microenvironment for treating tumors, and thus will be less likelyto cause side effects. In the embodiment where the biologic protein isan antibody against an antigen on the surface of the tumor cells wherethe antigen is exposed to the tumor microenvironment, the conditionallyactive antibody has lower affinity to the antigen than the wild-typeantibody in other parts of the body, e.g. a non-tumor microenvironment,while it has higher affinity to the antigen than the wild-type antibodyin the tumor microenvironment. Such conditionally active antibodies canbind weakly or not at all to the antigen in other parts of the body, buthave greater binding, or bind strongly and tightly, to the antigen inthe tumor microenvironment.

In some embodiments, the conditionally active antibody is an antibodyagainst an immune checkpoint protein, resulting in inhibition of theimmune checkpoints. Such conditionally active antibodies have at leastone of (1) an increased binding affinity to the immune checkpointprotein in a tumor microenvironment in comparison to the wild-typeantibody from which the conditionally active antibody is derived, and,(2) a decreased binding affinity to the immune checkpoint protein in anon-tumor microenvironment in comparison to the wild-type antibody fromwhich the conditionally active antibody is derived.

The immune checkpoints function as endogenous inhibitory pathways forthe immune system to maintain self-tolerance and modulate the durationand extent of immune response to antigenic stimulation, i.e., foreignmolecules, cells and tissues See Pardoll, Nature Reviews Cancer, vol.12, pages 252-264, 2012. Inhibition of immune checkpoints by suppressingone or more checkpoint proteins can cause super-activation of the immunesystem, especially T-cells, thus inducing the immune system to attacktumors. Checkpoint proteins suitable for the present invention includeCTLA4 and its ligands CD80 and CD86, PD1 and its ligands PDL1 and PDL2,T cell immunoglobulin and mucin protein-3 (TIM3) and its ligand GALS, Band T lymphocyte attenuator (BTLA) and its ligand HVEM (herpesvirusentry mediator), receptors such as killer cell immunoglobulin-likereceptor (KIR), lymphocyte activation gene-3 (LAGS) and adenosine A2areceptor (A2aR), as well as ligands B7-H3 and B7-H4. Additional suitableimmune checkpoint proteins are described in Pardoll, Nature ReviewsCancer, vol. 12, pages 252-264, 2012 and Nirschl & Drake, Clin CancerRes, vol. 19, pages 4917-4924, 2013.

CTLA-4 and PD1 are two of the best known immune checkpoint proteins.CTLA-4 can down-regulate pathways of T-cell activation (Fong et al.,Cancer Res. 69(2):609-615, 2009; and Weber, Cancer Immunol. Immunother,58:823-830, 2009). Blockading CTLA-4 has been shown to augment T-cellactivation and proliferation. Inhibitors of CTLA-4 include anti-CTLA-4antibodies. Anti-CTLA-4 antibodies bind to CTLA-4 and block theinteraction of CTLA-4 with its ligands CD80 or CD86 thereby blocking thedown-regulation of the immune responses elicited by the interaction ofCTLA-4 with its ligand.

The checkpoint protein PD1 is known to suppress the activity of T cellsin peripheral tissues at the time of an inflammatory response toinfection and to limit autoimmunity. An in vitro PD1 blockade canenhance T-cell proliferation and cytokine production in response tostimulation by specific antigen targets or by allogeneic cells in mixedlymphocyte reactions. A strong correlation between PD1 expression andreduced immune response was shown to be caused by the inhibitoryfunction of PD1, i.e., by inducing immune checkpoints (Pardoll, NatureReviews Cancer, 12: 252-264, 2012). A PD1 blockade can be accomplishedby a variety of mechanisms including antibodies that bind PD1 or itsligands, PDL1 or PDL2.

Past research has discovered antibodies against several checkpointproteins (CTLA4, PD1, PD-L1). These antibodies are effective in treatingtumors by inhibiting the immune checkpoints thereby super-activating theimmune system, especially the T-cells, for attacking tumors (Pardoll,Nature Reviews Cancer, vol. 12, pages 252-264, 2012). However, thesuper-activated T-cells may also attack host cells and/or tissues,resulting in collateral damage to a patient's body. Thus, therapy basedon use of these known antibodies for inhibition of immune checkpoints isdifficult to manage and the risk to the patient is a serious concern.For example, an FDA approved antibody against CTLA-4 carries a black boxwarning due to its high toxicity.

The present invention addresses the problem of collateral damage bysuper-activated T-cells by providing conditionally active antibodiesagainst immune checkpoint proteins. These conditionally activeantibodies preferentially activate the immune checkpoints in atumor-microenvironment. At the same time, the immune checkpoints in thenon-tumor-microenvironment(s), e.g. normal body tissue, are notinhibited or are less inhibited by the conditionally active antibodiessuch that in the non-tumor microenvironment the potential for collateraldamage to the body is reduced. This goal is achieved by engineering theconditionally active antibody to be more active in the tumormicroenvironment than in the non-tumor microenvironment.

In some embodiments, the conditionally active antibody against an immunecheckpoint protein may have a ratio of binding activity to an immunecheckpoint protein in the tumor-microenvironment to the binding activityto the same immune checkpoing protein in a non-tumor microenvironment ofat least about 1.1, or at least about 1.2, or at least about 1.4, or atleast about 1.6, or at least about 1.8, or at least about 2, or at leastabout 2.5, or at least about 3, or at least about 5, or at least about7, or at least about 8, or at least about 9, or at least about 10, or atleast about 15, or at least about 20. A typical assay for measuring thebinding activity of an antibody is an ELISA assay.

Highly immunogenic tumors, such as malignant melanoma, are mostvulnerable to a super-activated immune system achieved by immune systemmanipulation. Thus the conditionally active antibodies against immunecheckpoint proteins may be especially effective for treating such highlyimmunogenic tumors. However, other types of tumors are also vulnerableto a super-activated immune system.

In some embodiments, the conditionally active antibodies against theimmune checkpoint proteins may be used in combination therapy. Forexample, combination therapy may include a conditionally active antibodyagainst a tumor cell surface molecule (tumor specific antigen) and aconditionally active antibody against an immune checkpoint protein. Inone embodiment, both the binding activity of the conditionally activeantibody to the tumor cell surface molecule and the binding activity ofthe conditionally active antibody to the immune checkpoint protein mayreside in a single protein, i.e., a bispecific conditionally activeantibody as disclosed herein. In some further embodiments, combinationtherapy may include a conditionally active antibody against a tumor cellsurface molecule (tumor specific antigen) and two or more conditionallyactive antibodies against two or more different immune checkpointproteins. In one embodiment, all of these binding activities may residein a single protein, i.e., a multispecific antibody as disclosed herein.

Since the conditionally active antibodies are more active in a tumormicroenvironment in comparison with the activity of the wild-typeantibody against the same tumor cell surface molecule or checkpointprotein from which the conditionally active antibody is derived, thesecombination therapies can provide both an enhanced efficacy and asignificant reduction in toxicity. The reduced toxicity of theseconditionally active antibodies, especially the antibodies against theimmune checkpoint proteins, can allow safe use of potent antibodies,such as ADC antibodies as described herein, as well as a higher dose ofthe antibodies.

In some embodiments, the conditionally active antibodies against thecheckpoint proteins may be in a prodrug form. For example, theconditionally active antibodies may be prodrugs that have no desireddrug activity before being cleaved and turned into a drug form. Theprodrugs may be cleaved preferentially in a tumor-microenvironment,either because the enzyme that catalyzes such cleavage existspreferentially in the tumor-microenvironment or because theconditionally active antibodies make the cleavage site more accessiblein a tumor microenvironment, in comparison with the accessibility of thecleavage site in a non-tumor microenvironment.

Conditionally Active Biologic Proteins for Stem Cell Niches, IncludingTumor Stem Cells

Stem cells exist in an environment called stem cell niche in the body,which constitutes a basic unit of tissue physiology, integrating signalsthat mediate the response of stem cells to the needs of organisms. Yetthe niche may also induce pathologies by imposing aberrant functions onstem cells or other targets. The interplay between stem cells and theirniches creates the dynamic system necessary for sustaining tissues, andfor the ultimate design of stem-cell therapeutics (Scadden, “Thestem-cell niche as an entity of action,” Nature, vol. 441, pages1075-1079, 2006). Common stem cell niches in vertebrates include thegermline stem cell niche, the hematopoietic stem cell niche, the hairfollicle stem cell niche, the intestinal stem cell niche, and thecardiovascular stem cell niche.

The stem cell niche is a specialized environment that is different fromother parts of the body (e.g. blood plasma) (Drummond-Barbosa, “StemCells, Their Niches and the Systemic Environment: An Aging Network,”Genetics, vol. 180, pages 1787-1797, 2008; Fuchs, “Socializing with theNeighbors: Stem Cells and Their Niche,” Cell, vol. 116, pages 769-778,2004). The stem cell niche is hypoxic where oxidative DNA damage isreduced. Direct measurements of oxygen levels have revealed that bonemarrow is, in general, quite hypoxic (˜1%-2% O2), in comparison to bloodplasma (Keith et al., “Hypoxia-Inducible Factors, Stem Cells, andCancer,” Cell, vol. 129, pages 465-472, 2007; Mohyeldin et al., “Oxygenin Stem Cell Biology: A Critical Component of the Stem Cell Niche,” CellStem Cell, vol. 7, pages 150-161, 2010). In addition, the stem cellniches need to have several other factors to regulate stem cellcharacteristics within the niches: extracellular matrix components,growth factors, cytokines, and factors of the physiochemical nature ofthe environment including the pH, ionic strength (e.g. Ca²⁺concentration) and metabolites.

Accordingly, the stem cell niche has at least several physiologicalconditions that are different from those of the other parts of body,such as the physiological conditions in the blood plasma. The stem cellniche has a lower oxygen concentration (1-2%) than other parts of thebody, especially the blood plasma. Other physiological conditions forthe stem cell niche including pH and ionic strength, may also bedifferent from other parts of the body.

Stem cell therapy is an interventional strategy that introduces newadult stem cells into damaged tissue in order to treat disease orinjury. This strategy depends on the ability of stem cells to self-renewand give rise to subsequent offspring with variable degrees ofdifferentiation capacities. Stem cell therapy offers significantpotential for regeneration of tissues that can potentially replacediseased and damaged areas in the body, with minimal risk of rejectionand side effects. Therefore, delivering a drug (biologic protein (e.g.antibody) or chemical compound) to the stem cell niche for influencingthe renewal and differentiation of stem cells is an important part ofstem cell therapy.

There are several examples on how the stem cell niches influence therenewal and/or differentiation of the stem cells in mammals. The firstis in the skin, where the β-1 integrin is known to be differentiallyexpressed on primitive cells and to participate in constrainedlocalization of a stem-cell population through interaction with matrixglycoprotein ligands. Second, in the nervous system, the absence oftenascin C alters neural stem-cell number and function in thesubventricular zone. Tenascin C seems to modulate stem-cell sensitivityto fibroblast growth factor 2 (FGF2) and bone morphogenetic protein 4(BMP4), resulting in increased stem-cell propensity. Third, anothermatrix protein, the Arg-Gly-Asp-containing sialoprotein, osteopontin(OPN), has now been demonstrated to contribute to haematopoietic stemcell regulation. OPN interacts with several receptors known to be onhaematopoietic stem cells, CD44, and α4 and α5β1 integrins. OPNproduction can vary markedly, particularly with osteoblast activation.Animals deficient in OPN have an increased HS-cell number, because alack of OPN leads to superphysiologic stem-cell expansion understimulatory conditions. Therefore, OPN seems to serve as a constraint onhaematopoietic stem cell numbers, limiting the number of stem cellsunder homeostatic conditions or with stimulation. See Scadden, “Thestem-cell niche as an entity of action,” Nature, vol. 441, pages1075-1079, 2006.

Xie et al. “Autocrine signaling based selection of combinatorialantibodies that transdifferentiate human stem cells,” Proc Natl Acad SciUSA, vol. 110, pages 8099-8104, 2013) discloses a method of usingantibodies to influence stem cell differentiation. The antibodies areagonists for a granulocyte colony stimulating factor receptor. Unlikethe natural granulocyte-colony stimulating factor that activates cellsto differentiate along a predetermined pathway, the isolated agonistantibodies transdifferentiated human myeloid lineage CD34+ bone marrowcells into neural progenitors. Melidoni et al. (“Selecting antagonisticantibodies that control differentiation through inducible expression inembryonic stem cells,” Proc Natl Acad Sci USA, vol. 110, pages17802-17807, 2013) also discloses a method of using an antibody tointerfere the interaction between FGF4 and its receptor FGFR1β,therefore block the autocrine FGF4-mediated embryonic stem celldifferentiation.

Knowledge of the functions of ligands/receptors in stem celldifferentiation has enabled the strategy of applying biologic proteinsto interfere with these ligands/receptors for the purpose of regulatingor even directing stem cell differentiation. The ability to controldifferentiation of genetically unmodified human stem cells through theadministration of antibodies into the stem cell niche can provide new exvivo or in vivo approaches to stem cell-based therapeutics. In someembodiments, the present invention provides a conditionally activebiologic protein generated from a wild-type biologic protein that iscapable of entering the stem cell niches, including cancer stem cells,to regulate stem cell or tumor development. The conditionally activebiologic protein has lower activity than the wild-type biologic proteinunder at least one physiological condition in other parts of the body,while it has higher activity than the wild-type biologic protein underat least one physiological condition in the stem cell niche, for examplethe cancer stem cell environment. Such conditionally active biologicproteins will be less likely to cause side effects and preferentiallyact in the stem cell niche to regulate renewal and differentiation ofstem cells. In some embodiments, the conditionally active biologicproteins are antibodies. Such conditionally active antibodies can bindweakly or not at all to their antigens in other parts of the body, butbind strongly and tightly to the antigens in the stem cell niche.

The conditionally active biologic proteins for the synovial fluid, tumormicroenvironment and stem cell niches of the present invention aregenerated by a method for evolving a DNA that encodes a wild-typebiologic protein to create a mutant DNA library. The mutant DNA libraryis then expressed to obtain mutant proteins. The mutant proteins arescreened for a conditionally active biologic protein that has a higheractivity than the wild-type biologic protein under at least onephysiological condition of a first part of the body selected from thegroup consisting of synovial fluid, tumor microenvironment, and stemcell niches, and has lower activity than the wild-type biologic proteinunder at least one physiological condition at a second part of the bodythat is different from the first part of the body. The second part ofthe body may be the blood plasma. Such selected mutant biologic proteinsare conditionally active biologic proteins that have high activity inthe first part of the body but low activity in the second parts of thebody.

Such conditionally active biologic proteins are advantageous in loweringside effects of the wild-type protein, since the conditionally activebiologic protein has lower activity in the other parts of the body wherethe conditionally active biologic protein is not intended to act. Forinstance, if the conditionally active biologic protein is intended to beintroduced into the tumor microenvironment, the fact that theconditionally active biologic protein has low activity in parts of thebody other than the tumor microenvironment means such conditionallyactive biologic protein will be less likely to interfere with normalphysiological functions in parts of the body other than the tumormicroenvironment. At the same time, the conditionally active biologicprotein has high activity in the tumor microenvironment, which gives theconditionally active biologic protein a higher efficacy in treatingtumors.

Because of the reduced side effects, the conditionally active biologicprotein will allow a significantly higher dose of the protein to besafely used, in comparison with the wild-type biologic protein. This isespecially beneficial for an antibody against a cytokine or a growthfactor, because antibodies against the cytokine or growth factor mayinterfere with normal physiological functions of the cytokine or growthfactor in other parts of the body. By using a conditionally activebiologic protein, with reduced side effects, higher doses may be used toachieve higher efficacy.

The conditionally active biologic proteins for acting in one of asynovial fluid, tumor microenvironment, or stem cell niche can alsoenable new drug targets to be used. Using traditional biologic proteinsas therapeutics may cause unacceptable side effects. For example,inhibition of an epidermal growth factor receptor (EGFR) can veryeffectively suppress tumor growth. However, a drug inhibiting EGFR willalso suppress growth at the skin and gastrointestinal (GI) tract. Theside effects render EGFR unsuitable as a tumor drug target. Using aconditionally active antibody that binds to EGFR at high affinity inonly the tumor microenvironment, but not or at very low affinity at anyother parts of the body, will significantly reduce the side effects andat the same time suppress tumor growth. In this case, EGFR may become aneffective new tumor drug target by using conditionally activeantibodies.

In another example, suppressing cytokines is often beneficial inrepairing joint damage. However, suppressing cytokines in other parts ofthe body also may suppress the immune response of the body, causing animmune deficiency. Thus, cytokines in synovial fluid are not idealtargets for developing traditional antibody drugs for treatment of jointdamage. However, by using conditionally active antibodies thatpreferentially bind to cytokines in the synovial fluid, while not oronly weakly to the same cytokines in other parts of the body, the sideeffect of immune deficiency can be dramatically reduced. Therefore,cytokines in synovial fluid may become suitable targets for repairingjoint damage by using conditionally active antibodies.

Conditionally Active Biologic Proteins for Organs/Tissues Susceptible toInflammation

In some embodiments, the conditionally active biologic proteins aredesigned to preferentially act in organs or tissues that are susceptibleto inflammation, such as a lymph node, a tonsil, an adenoid, and asinus. Additional organs and tissues that are susceptible toinflammation may be found in anatomy textbooks such as Gray's Anatomy byHenry Gray, 41^(st) edition, 2015, published by Elsevier.

These organs and tissues typically exhibit at least one aberrantcondition once they are inflamed. For example, these inflamed organs andtissues may have higher osmotic pressure and/or a lower concentration ofone or more ions, in comparison with, for example, the normalphysiological conditions of other parts of the body such as human bloodplasma. Further, there may be higher concentrations of small molecules,lactic acid, cytokines and white blood cells in such inflamed organs andtissues as compared to the normal physiological conditions of otherparts of the body such as human blood plasma.

In some embodiments, the conditionally active biologic proteins may beproduced by the present invention using an aberrant condition selectedfrom one or more aberrant conditions encountered in an area ofinflammation and a normal physiological condition in the human bloodplasma. Such conditionally active biologic proteins would thus have ahigher activity in the organs/tissues in an inflammatory state than theactivity of the wild-type biologic protein and lower activity in humanblood plasma than the activity of the wild-type biologic protein. Suchconditionally active biologic proteins can preferentially act in aninflamed region of the body, but will have little or no activity in aregion of the body that is not inflamed.

Conditionally Active Viral Particles

Viral particles have long been used as delivery vehicles fortransporting proteins, nucleic acid molecules, chemical compounds orradioactive isotopes to a target cell or tissue. Viral particles thatare commonly used as delivery vehicles include retoviruses,adenoviruses, lentivirus, herpes virus, and adeno-associated viruses.The viral particles recognize their target cells through a surfaceprotein that serves as a recognition protein for specific binding to acellular protein that serves as target protein of the target cells,often in a ligand-receptor binding system (Lentz, “The reognition eventbetween virus and host cell receptor: a target for antiviral agents,” J.of Gen. Virol., vol. 71, pages 751-765, 1990). For example, the viralrecognition protein may be a ligand for a receptor on the target cells.The specificity between a ligand and a receptor allows the viralparticles to specifically recognize and deliver their content to atarget cell.

Techniques for developing artificial viral particles from wild-typeviruses are well known to a person skilled in the art. Known artificialviral particles as delivery vehicles include these based on retroviruses(see, e.g., WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; U.S.Pat. No. 5,219,740; WO 93/11230; WO 93/1.0218; U.S. Pat. No. 4,777,127;GB Patent No. 2,200,651; EP 0 345 242; and WO 91/02805), alphavirus(e.g., Sindbis virus vectors, Semliki forest virus (ATCC VR-67; ATCCVR-1247), Ross River virus (ATCC VR-373; ATCC. VR-1246), Venezuelanequine encephalitis virus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCCVR-532)), and adeno-associated viruses (see, e.g., WO 94/12649, WO93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655).

Generally, the artificial viral particles are constructed by inserting aforeign recognition protein into a virus particle, often replacing thenative recognition protein by recombinant technology. The foreignrecognition protein may be, for example, an antibody, a receptor, aligand or a collagen binding domain. The present invention provides aconditionally active recognition protein that is inactive or less activefor binding to a cell at a normal physiological condition, and that isactive or more active for binding to a cell at an aberrant condition.The conditionally active recognition protein can thereby preferentiallybind to target cells of diseased tissue and/or at a disease site basedon the presence of an abnormal condition at that site and avoid or onlyminimally bind to the cells of normal tissue where a normalphysiological condition exists. The conditionally active recognitionprotein may be expressed and displayed on the surface of a viralparticle.

In some embodiments, the present invention provides a method of evolvinga wild-type recognition protein and screening for a conditionally activerecognition protein. The conditionally active recognition protein isless active in binding to a cell than the wild-type recognition proteinunder a normal physiological condition, and more active in binding to acell than the wild-type recognition protein under an aberrant condition.Such a conditionally active recognition protein may be inserted into aviral particle by well-known recombinant technology to generate aconditionally active viral particle.

In another embodiment, the present invention provides a conditionallyactive viral particle including a conditionally active recognitionprotein, which allows the conditionally active viral particle torecognize and bind with the target cells of diseased tissue or at adisease site, but not the cells of normal tissue. Such a conditionallyactive viral particle can preferentially deliver therapeutics within theviral particle to the disease tissue or disease site, while theconditionally active viral particle delivers less or does not deliverthe therapeutics to the cells of normal tissue.

In some embodiments, the target cells at a disease site are inside azone or microenvironment with an abnormal pH (e.g., pH 6.5) or anabnormal temperature, in comparison with the pH or temperature in otherparts of the body that are healthy or not suffering from the particulardisease or disease state. In this embodiment, the conditionally activerecognition protein is less active than a wild-type recognition proteinin binding with a target protein of a target cell at a normalphysiological pH or temperature, and more active than a wild-typerecognition protein in binding with the target protein of a target cellat an abnormal pH or temperature. In this manner, the recognitionprotein will preferentially bind at a site where an abnormal pH ortemperature is encountered thereby delivering a treatment to the site ofa disease.

In one embodiment, the viral particle may include a conditionally activeantibody of the present invention, and especially the variable region ofan antibody (e.g., Fab, Fab′, Fv). Such a conditionally active antibodycan bind to the target protein (as antigen) of a target cell with loweraffinity than a wild-type antibody under a normal physiologicalcondition which may be encountered at a location with normal tissue, anda higher affinity than the wild-type antibody under aberrant conditionwhich may be encountered at a disease site or diseased tissue. Theconditionally active antibody may be derived from the wild-type antibodyaccording to the method of the present invention.

In an embodiment, the target protein on the target cell includestyrosine kinase growth factor receptors which are overexpressed on thecell surfaces in, for example, many tumors. Exemplary tyrosine kinasegrowth factors are VEGF receptors, FOP receptors, PDGF receptors, IGFreceptors, EGF receptors, TGF-alpha receptors, TOP-beta receptors,HB-EGF receptors, ErbB2 receptors, ErbB3 receptors, and ErbB4 receptors.

Conditionally Active DNA/RNA Modifying Proteins

DNA/RNA modifying proteins have been discovered as a form of newgenome-engineering tools, particularly one called CRISPR, which canallow researchers to perform microsurgery on genes, precisely and easilychanging a DNA sequence at exact locations on a chromosome (genomeediting, Mali et al., “Cas9 as a versatile tool for engineeringbiology,” Nature Methods, vol. 10, pages 95′7-963, 2013). For example,sickle-cell anemia is caused by a single base mutation, which canpotentially be corrected using DNA/RNA modifying proteins. Thetechnology may precisely delete or edit bits of a chromosome, even bychanging a single base pair (Makarova et al., “Evolution andclassification of the CRISPR-Cas systems,” Nature Reviews Microbiology,vol. 9, pages 467-477, 2011).

Genome editing with CRISPR has the ability to quickly and simultaneouslymake multiple genetic changes to a cell. Many human illnesses, includingheart disease, diabetes, and neurological diseases, are affected bymutations in multiple genes. This CRISPR-based technology has thepotential to reverse the disease causing mutations and cure thesediseases or at least reduce the severity of these diseases. Genomeediting relies on CRISPR associated (Cas) proteins (a family of enzymes)for cutting the genomic DNA. Typically, the Cas protein is guided by asmall guide RNA to a targeted region in the genome, where the guide RNAmatches the target region. Because the Cas protein has little or nosequence specificity, the guide RNA serves as a pointer for the Casprotein to achieve precise genome editing. In one embodiment, one Casprotein may be used with multiple guide RNAs to simultaneously correctmultiple gene mutations.

There are many Cas proteins. Examples include Cas1, Cas2, Cas3′, Cas3″,Cas4, Cas5, Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c,Cas9, Cas10, Cas10d, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5,Csn2, Csm2, Csmn3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1,Csb2, Csb3, Csx17, Csx1.4, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1,Csf2, Csf3, and Csf4 ((Makarova et al., “Evolution and classification ofthe CRISPR-Cas systems,” Nature Reviews Microbiology, vol. 9, pages467-477, 2011).

To conduct genome editing, the Cas protein has to enter the target cell.Cells in a subject may have a different intracellular pH inside of thecells. Some cells in diseased tissue have an abnormal intracellular pH.For example, some tumor cells tend to have an alkaline intracellular pHof about 7.12-7.65, while cells in normal tissue have a neutralintracellular pH ranging from 6.99-7.20. See Cardone et al., “The roleof disturbed pH dynamics and the Na(+)/H(+) exchanger in metatasis,”Nat. Rev. Cancer, vol. 5, pages 786-795, 2005. In chronic hypoxia, thecells in diseased tissue have an intracellular pH of about 7.2-7.5, alsohigher than the intracellular pH of normal tissue (Rios et al., “Chronichypoxia elevates intracellular pH and activates Na+/H+ exchange inpulmonary arterial smooth muscle cells,” American Journal ofPhysiology—Lung Cellular and Molecular Physiology, vol. 289, pagesL867-L874, 2005). Further, in ischemia cells, the intracellular pH istypical in a range of 6.55-6.65, which is lower than the intracellularpH of normal tissue (Haqberg, “Intracellular pH during ischemia inskeletal muscle: relationship to membrane potential, extracellular pH,tissue lactic acid and ATP,” Pflugers Arch., vol. 404, pages 342-347,1985). More examples of abnormal intracellular pH in diseased tissue arediscussed in Han et al., “Fluorescent Indicators for Intracellular pH,”Chem Rev., vol. 110, pages 2709-2728, 2010.

The present invention provides a method for producing a conditionallyactive Cas protein from a wild-type Cas protein, where the conditionallyactive Cas protein has at least one of (1) a decreased enzymaticactivity relative to the activity of the wild-type Cas protein under anormal physiological condition inside a normal cell, and (2) anincreased enzymatic activity relative to the activity of the wild-typeCas protein under an aberrant condition inside a target cell such as oneof the diseased cells discussed above. In some embodiments, the normalphysiological condition is an intracellular pH about neutral, and theaberrant condition is a different intracellular pH that is above orbelow neutral. In an embodiment, the aberrant condition is anintracellular pH of from 7.2 to 7.65 or an intracellular pH of from6.5-6.8.

In some embodiments, the conditionally active Cas protein may bedelivered to a target cell using the conditionally active viral particleof the present invention. The conditionally active viral particleincludes the conditionally active Cas protein and at least one guide RNAfor directing the Cas protein to the location at which Cas protein willedit the genomic DNA.

Multispecific antibodies have high selectivity at preferentiallytargeting tissues containing all or most of the targets (antigens) thata multispecific antibody can bind to. For example, a bispecific antibodyprovides selectivity for target cells by displaying greater preferenceto target cells that express both of the antigens recognized by thebispecific antibody, in comparison with non-target cells that mayexpress only one of the antigens. Therefore, due to the dynamism of thesystem, there are more bispecific antibodies being bound to the targetcells than non-target cells at equilibrium.

The multispecific antibodies engineered herein, or theirantigen-recognition fragments, may be used as the ASTR in the chimericantigen receptor of the present invention.

Engineering Cytotoxic Cells

Once a conditionally active ASTR is identified by the screening step,the chimeric antigen receptor may be assembled by ligating thepolynucleotide sequences encoding the individual domains to form asingle polynucleotide sequence (the CAR gene, which encodes theconditionally active CAR). The individual domains include aconditionally active ASTR, a TM, and an ISD. In some embodiments, otherdomains may also be introduced in the CARs, including an ab ESD and aCSD (FIG. 1). If the conditionally active CAR is a bispecific CAR, theCAR gene may be, for example, in the following configuration in theN-terminal to C-terminal direction: N-terminal signal sequence-ASTR1-linker-ASTR 2-extracellular spacer domain-transmembranedomain-co-stimulatory domain-intracellular signaling domain. In oneembodiment, such a CAR gene may include two or more co-stimulatorydomains.

Alternatively, the polynucleotide sequence encoding the conditionallyactive CAR may be in the following configuration in the N-terminal toC-terminal direction: N-terminal signal sequence-ASTR 1-linker-ASTR2-transmembrane domain-co-stimulatory domain-intracellular signalingdomain. In an embodiment, such a CAR may include two or moreco-stimulatory domains. If a CAR includes more than two ASTRs, thepolynucleotide sequence encoding the CAR may be in the followingconfiguration in the N-terminal to C-terminal direction: N-terminalsignal sequence-ASTR 1-linker-ASTR 2-linker-(antigen-specific targetingregion)_(n)-transmembrane domain-co-stimulatory domain-intracellularsignaling domain. Such a CAR may further include an extracellular spacerdomain. Each ASTR may be separated by a linker. In an embodiment, such aCAR may include two or more co-stimulatory domains.

The conditionally active CAR is introduced into the cytotoxic cells byan expression vector. Expression vectors including a polynucleotidesequence encoding a conditionally active CAR of the invention are alsoprovided herein. Suitable expression vectors include lentivirus vectors,gamma retrovirus vectors, foamy virus vectors, adeno associated virus(AAV) vectors, adenovirus vectors, engineered hybrid viruses, naked DNA,including but not limited to transposon mediated vectors, such asSleeping Beauty, Piggybak, and Integrases such as Phi31. Some othersuitable expression vectors include Herpes simplex virus (HSV) andretrovirus expression vectors.

Adenovirus expression vectors are based on adenoviruses, which have alow capacity for integration into genomic DNA but a high efficiency fortransfecting host cells. Adenovirus expression vectors containadenovirus sequences sufficient to: (a) support packaging of theexpression vector and (b) to ultimately express the CAR gene in the hostcell. The adenovirus genome is a 36 kb, linear, double stranded DNA,where a foreign DNA sequence (such as CAR genes) may be inserted tosubstitute large pieces of adenoviral DNA in order to make theexpression vector of the present invention (Grunhaus and Horwitz,“Adenoviruses as cloning vectors,” Seminars Virol., vol. 3, pages237-252, 1992).

Another expression vector is based on an adeno associated virus, whichtakes advantage of the adenovirus coupled systems. This AAV expressionvector has a high frequency of integration into the host genome. It caneven infect nondividing cells, thus making it useful for delivery ofgenes into mammalian cells, for example, in tissue cultures or in vivo.The AAV vector has a broad host range for infectivity. Detailsconcerning the generation and use of AAV vectors are described in U.S.Pat. Nos. 5,139,941 and 4,797,368.

Retrovirus expression vectors are capable of integrating into the hostgenome, delivering a large amount of foreign genetic material, infectinga broad spectrum of species and cell types and being packaged in specialcell lines. The retrovirus vector is constructed by inserting a nucleicacid (e.g., one encoding the CAR) into the viral genome at certainlocations to produce a virus that is replication defective. Though theretrovirus vectors are able to infect a broad variety of cell types,integration and stable expression of the CAR gene requires the divisionof host cells.

Lentivirus vectors are derived from lentiviruses, which are complexretroviruses that, in addition to the common retroviral genes gag, pol,and env, contain other genes with regulatory or structural function(U.S. Pat. Nos. 6,013,516 and 5,994,136). Some examples of lentivirusesinclude the Human Immunodeficiency Viruses (HIV-1, HIV-2) and the SimianImmunodeficiency Virus (SIV). Lentivirus vectors have been generated bymultiply attenuating the HIV virulence genes, for example, the genesenv, vif, vpr, vpu and nef are deleted making the vector biologicallysafe. Lentivirus vectors are capable of infecting non-dividing cells andcan be used for both in vivo and ex vivo gene transfer and expression ofthe CAR gene (U.S. Pat. No. 5,994,136).

Expression vectors including the conditionally active CAR gene can beintroduced into a host cell by any means known to person skilled in theart. The expression vectors may include viral sequences fortransfection, if desired. Alternatively, the expression vectors may beintroduced by fusion, electroporation, biolistics, transfection,lipofection, or the like. The host cell may be grown and expanded inculture before introduction of the expression vectors, followed by theappropriate treatment for introduction and integration of the vectors.The host cells are then expanded and screened by virtue of a markerpresent in the vectors. Various markers that may be used include hprt,neomycin resistance, thymidine kinase, hygromycin resistance, etc. Asused herein, the terms “cell,” “cell line,” and “cell culture” may beused interchangeably. In some embodiments, the host cell is a T cell, NKcell and NKT cell.

In another aspect, the present invention also provides geneticallyengineered cytotoxic cells which include and stably express theconditionally active CAR of the invention. In one embodiment, thegenetically engineered cells include T-lymphocytes (T cells), naive Tcells (T_(N)), memory T cells (for example, central memory T cells(T_(CM)), effector memory cells (T_(EM))), natural killer cells, andmacrophages capable of giving rise to therapeutically relevant progeny.In another embodiment, the genetically engineered cells are autologouscells. Examples of suitable T cells include CD4⁺/CD8⁻, CD4⁻/CD8⁺,CD4⁻/CD8⁻ or CD4⁺/CD8⁺ T cells. The T cells may be a mixed population ofCD4⁺/CD8⁻ and CD4⁻/CD8⁺ cells or a population of a single clone. CD4⁺ Tcells of the invention may also produce IL-2, IFN-gamma, TNF-alpha andother T cell effector cytokines when co-cultured in vitro with cellsexpressing the target antigens (for example CD20⁺ and/or CD 19⁺ tumorcells). CD8⁺ T cells of the invention may lyse cells expressing thetarget antigen. In some embodiments, T cells may be any one or more ofCD45RA⁺ CD62L⁺ naive cells, CD45RO CD62I7 central memory cells, CD62L⁻effector memory cells or a combination thereof (Berger et al., “Adoptivetransfer of virus-specific and tumor-specific T cell immunity,” Curr.Opin. Immunol., vol. 21, pages 224-232, 2009).

Genetically engineered cytotoxic cells may be produced by stablytransfecting host cells with an expression vector including the CAR geneof the invention. Additional methods to genetically engineer thecytotoxic cells using the expression vector include chemicaltransformation methods (e.g., using calcium phosphate, dendrimers,liposomes and/or cationic polymers), non-chemical transformation methods(e.g., electroporation, optical transformation, gene electrotransferand/or hydrodynamic delivery) and/or particle-based methods (e.g.,impalefection, using a gene gun and/or magnetofection). Transfectedcells demonstrating the presence of a single integrated un-rearrangedvector and expressing the conditionally active CAR may be expanded exvivo.

Physical methods for introducing an expression vector into host cellsinclude calcium phosphate precipitation, lipofection, particlebombardment, microinjection, electroporation, and the like. Methods forproducing cells including vectors and/or exogenous nucleic acids arewell-known in the art. See, for example, Sambrook et al. (2001,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,New York). Chemical methods for introducing an expression vector into ahost cell include colloidal dispersion systems, such as macromoleculecomplexes, nanocapsules, microspheres, beads, and lipid-based systemsincluding oil-in-water emulsions, micelles, mixed micelles, andliposomes.

After the expression vector containing the CAR gene is introduced intothe host cells, the CAR gene will be expressed thus producing a CARmolecule that can bind to the target antigen. The produced CAR moleculebecomes a transmembrane protein by virtue of having a transmembranedomain. The host cells will then be converted to CAR cells such as CAR-Tcells. The process for producing engineered cytotoxic cells with the CARmolecule, for example CAR-T cells, has been described in, for example,(Cartellieri et al., “Chimeric antigen receptor-engineered T cells forimmunotherapy of cancer,” Journal of Biomedicine and Biotechnology, vol.2010, Article ID 956304, 2010; and Ma et al., “Versatile strategy forcontrolling the specificity and activity of engineered T cells,” PNAS,vol. 113, E450-E458, 2016).

Whether prior to or after genetic modification of the cytotoxic cells toexpress a desirable conditionally active CAR, the cells can be activatedand expanded in number using methods as described, for example, in U.S.Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358;6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566;7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and US20060121005. For example, the T cells of the invention may be expandedby contact with a surface having attached thereto an agent thatstimulates a CD3/TCR complex associated signal and a ligand thatstimulates a co-stimulatory molecule on the surface of the T cells. Inparticular, T cell populations may be stimulated by contact with ananti-CD3 antibody, or antigen-binding fragment thereof, or au anti-CD2antibody immobilized on a surface, or by contact with a protein kinase Cactivator (e.g., bryostatin) in conjunction with a calcium ionophore.For co-stimulation of an accessory molecule on the surface of the Tcells, a ligand that binds the accessory molecule is used. For example,T cells can be contacted with an anti-CD3 antibody and an anti-CD28antibody, under conditions appropriate for stimulating proliferation ofthe T cells. To stimulate proliferation of either CD4⁺ T cells or CD8⁺ Tcells, an anti-CD3 antibody and an anti-CD28 antibody. Examples of ananti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Besancon,France) and these can be used in the invention, as can other methodscommonly known in the art (Berg et al., Transplant Proc.30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9):13191328,1999; Garland et al., J. Immunol. Meth. 227(1-2):53-63, 1999).

In various embodiments, the present invention provides pharmaceuticalcompositions including a pharmaceutically acceptable excipient and atherapeutically effective amount of the conditionally active CAR of theinvention. The conditionally active CAR in the composition may be anyone or more of a polynucleotide encoding the CAR, a protein includingthe CAR or genetically modified cells expressing the CAR protein. TheCAR protein may be in the form of a pharmaceutically acceptable salt.Pharmaceutically acceptable salts refers to salts which can be used assalts of a therapeutic protein in the pharmaceutical industry, includingfor example, salts of sodium, potassium, calcium and the like, and aminesalts of procaine, dibenzylamine, ethylenediamine, ethanolamine,methylglucamine, taurine, and the like, as well as acid addition saltssuch as hydrochlorides, and basic amino acids and the like.

The pharmaceutically acceptable excipient may include any excipient thatis useful in preparing a pharmaceutical composition that is generallysafe, non-toxic, and desirable, and includes excipients that areacceptable for veterinary use as well as for human pharmaceutical use.Such excipients may be solid, liquid, semisolid, or, in the case of anaerosol composition, gaseous. One type of excipient includespharmaceutically acceptable carriers, which may be added to enhance orstabilize the composition, or to facilitate preparation of thecomposition. Liquid carriers include syrup, peanut oil, olive oil,glycerin, saline, alcohols and water. Solid carriers include starch,lactose, calcium sulfate, dihydrate, terra alba, magnesium stearate orstearic acid, talc, pectin, acacia, agar and gelatin. The carrier mayalso include a sustained release material such as glyceryl monostearateor glyceryl distearate, alone or with a wax.

The pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there are a widevariety of suitable formulations of pharmaceutical compositions of thepresent invention. A variety of aqueous carriers can be used, e.g.,buffered saline and the like. These solutions are sterile and generallyfree of undesirable matter. These compositions may be sterilized byconventional, well known sterilization techniques. The compositions maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions such as pH adjustment agents andbuffering agents, toxicity adjusting agents and the like, for example,sodium acetate, sodium chloride, potassium chloride, calcium chloride,sodium lactate and the like. The concentration of CAR in theseformulations can vary widely, and will be selected primarily based onfluid volumes, viscosities, and body weight in accordance with theparticular mode of administration selected and the patient's needs.

In various embodiments, the pharmaceutical compositions according to theinvention may be formulated for delivery via any suitable route ofadministration. “Route of administration” may refer to anyadministration pathway known in the art, including but not limited toaerosol, nasal, oral, intravenous, intramuscular, intraperitoneal,inhalation, transmucosal, transdermal, parenteral, implantable pump,continuous infusion, topical application, capsules and/or injections.

The pharmaceutical compositions according to the invention can beencapsulated, tableted or prepared in an emulsion or syrup for oraladministration. The pharmaceutical compositions are made following theconventional techniques of pharmacy involving milling, mixing,granulation, and compression, when necessary, for tablet forms; ormilling, mixing and filling for hard gelatin capsule forms. When aliquid carrier is used, the preparation may be in the form of syrup,elixir, emulsion or an aqueous or non-aqueous suspension. Such a liquidformulation may be administered directly p.o. or filled into a softgelatin capsule.

The pharmaceutical compositions may be formulated as: (a) liquidsolutions, such as an effective amount of the packaged nucleic acidsuspended in diluents, such as water, saline or PEG 400; (b) capsules,sachets or tablets, each containing a predetermined amount of the activeingredient, as liquids, solids, granules or gelatin; (c) suspensions inan appropriate liquid; and (d) suitable emulsions. Particularly,suitable dosage forms include, but are not limited to, tablets, pills,powder, dragees, capsules, liquids, lozenges, gels, syrups, slurries,suspensions, etc.

The solid formulations include suitable solid excipients such ascarbohydrates or protein fillers including, e.g., sugars such aslactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice,potato, or other plants; cellulose such as methyl cellulose,hydroxypropyhnethyl cellulose, and sodium carboxy-methylcellulose; andgums including arabic and tragacanth; and proteins, e.g., gelatin andcollagen. Disintegrating or solubilizing agents may be added, such ascross-linked polyvinyl pyrrolidone, agar, alginic acid, or a saltthereof, such as sodium alginate. Tablet forms can include one or moreof lactose, sucrose, mannitol, sorbitol, calcium phosphates, cornstarch, potato starch, tragacanth, microcrystalline cellulose, acacia,gelatin, colloidal silicon dioxide, croscannellose sodium, talc,magnesium stearate, stearic acid, and other excipients, colorants,fillers, binders, diluents, buffering agents, moistening agents,preservatives, flavoring agents, dyes, disintegrating agents, andpharmaceutically acceptable carriers.

The liquid suspensions include a conditionally active CAR, in admixturewith excipients suitable for the manufacture of aqueous suspensions.Such excipients include a suspending agent, such as sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia,and dispersing or wetting agents such as a naturally occurringphosphatide (e.g., lecithin), a condensation product of an alkyleneoxide with a fatty acid (e.g., polyoxyethylene stearate), a condensationproduct of ethylene oxide with a long chain aliphatic alcohol (e.g.,heptadecaethylene oxycetanol), a condensation product of ethylene oxidewith a partial ester derived from a fatty acid and a hexitol (e.g.,polyoxyethylene sorbitol mono-oleate), or a condensation product ofethylene oxide with a partial ester derived from fatty acid and ahexitol anhydride (e.g., polyoxyethylene sorbitan mono-oleate). Theliquid suspension can also contain one or more preservatives such asethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one ormore flavoring agents and one or more sweetening agents, such assucrose, aspartame or saccharin. Formulations can be adjusted forosmolality.

The lozenge forms can include the active ingredient in a flavor, usuallysucrose and acacia or tragacanth, as well as pastilles including theactive ingredient in an inert base, such as gelatin and glycerin orsucrose and acacia emulsions, gels, and the like containing, in additionto the active ingredient, carriers known in the art. It is recognizedthat the conditionally active CAR, when administered orally, must beprotected from digestion. This is typically accomplished either bycomplexing the conditionally active CAR with a composition to render itresistant to acidic and enzymatic hydrolysis or by packaging theconditionally active CAR in an appropriately resistant carrier such as aliposome. Means of protecting proteins from digestion are well known inthe art. The pharmaceutical compositions can be encapsulated, e.g., inliposomes, or in a formulation that provides for slow release of theactive ingredient.

The pharmaceutical composition may be formulated as aerosol formulations(e.g., they can be “nebulized”) to be administered via inhalation.Aerosol formulations can be placed into pressurized acceptablepropellants, such as dichlorodifluoromethane, propane, nitrogen, and thelike. Suitable formulations for rectal administration include, forexample, suppositories, which consist of the packaged nucleic acid witha suppository base. Suitable suppository bases include natural orsynthetic triglycerides or paraffin hydrocarbons, in addition, it isalso possible to use gelatin rectal capsules which consist of acombination of the packaged nucleic acid with a base, including, forexample, liquid triglycerides, polyethylene glycols, and paraffinhydrocarbons.

The pharmaceutical composition may be formulated for parenteraladministration, such as, for example, by intra-articular (in thejoints), intravenous, intramuscular, intradermal, intraperitoneal, andsubcutaneous routes, include aqueous and non-aqueous, isotonic sterileinjection solutions, which can contain antioxidants, buffers,bacteriostats, and solutes that render the formulation isotonic with theblood of the intended recipient, and aqueous and nonaqueous sterilesuspensions that can include suspending agents, solubilizers, thickeningagents, stabilizers, and preservatives, in the practice of thisinvention, compositions can be administered, for example, by intravenousinfusion, orally, topically, intraperitoneally, intravesically orintrathecally. In one aspect, parenteral modes of administration arepreferred methods of administration for compositions including the CARprotein or genetically engineered cytotoxic cells. The compositions mayconveniently be administered in unit dosage form and may be prepared byany of the methods well-known in the pharmaceutical art, for example asdescribed in Remington's Pharmaceutical Sciences, Mack Publishing Co.Easton Pa., 18^(th) Ed., 1990. Formulations for intravenousadministration may contain a pharmaceutically acceptable carrier such assterile water or saline, polyalkylene glycols such as polyethyleneglycol, oils of vegetable origin, hydrogenated naphthalenes and thelike.

The pharmaceutical composition may be administered by at least one modeselected from parenteral, subcutaneous, intramuscular, intravenous,intrarticular, intrabronchial, intraabdominal, intracapsular,intracartilaginous, intracavitary, intracelial, intracelebellar,intracerebroventricular, intracolic, intracervical, intragastric,intrahepatic, intramyocardial, intraosteal, intrapelvic,intrapericardiac, intraperitoneal, intrapleural, intraprostatic,intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal,intrasynovial, intrathoracic, intrauterine, intravesical, bolus,vaginal, rectal, buccal, sublingual, intranasal, or transdermal. Themethod can optionally further include administering, prior to,concurrently, or after the conditionally active CAR at least onecomposition including an effective amount of at least one compound orprotein selected from at least one of a detectable label or reporter, aTNF antagonist, an antirheumatic, a muscle relaxant, a narcotic, anon-steroid anti-inflammatory drug (NSAK)), an analgesic, an anesthetic,a sedative, a local anesthetic, a neuromuscular blocker, anantimicrobial, an antipsoriatic, a corticosteriod, an anabolic steroid,an erythropoietin, an immunization, an immunoglobulin, animmunosuppressive, a growth hormone, a hormone replacement drug, aradiopharmaceutical, an antidepressant, an antipsychotic, a stimulant,an asthma medication, a beta agonist, an inhaled steroid, an epinephrineor analog thereof, a cytotoxic or other anti-cancer agent, ananti-metabolite such as methotrexate, or an antiproliferative agent.

The types of cancers to be treated with the genetically engineeredcytotoxic cells or pharmaceutical compositions of the invention include,carcinoma, blastoma, and sarcoma, and certain leukemia or lymphoidmalignancies, benign and malignant tumors, and malignancies e.g.,sarcomas, carcinomas, and melanomas. The cancers may be non-solid tumors(such as hematological tumors) or solid tumors. Adult tumors/cancers andpediatric tumors/cancers are also included.

Hematologic cancers are cancers of the blood or bone marrow. Examples ofhematological (or hematogenous) cancers include leukemias, includingacute leukemias (such as acute lymphocytic leukemia, acute myelocyticleukemia, acute myelogenous leukemia and myeloblastic, promyelocytic,myelomonocytic, monocytic and erythroleukemia), chronic leukemias (suchas chronic myelocytic (granulocytic) leukemia, chronic myelogenousleukemia, and chronic lymphocytic leukemia), polycythemia vera,lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and highgrade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavychain disease, myelodysplastic syndrome, hairy cell leukemia andmyelodysplasia.

Solid tumors are abnormal masses of tissue that usually do not containcysts or liquid areas. Solid tumors can be benign or malignant.Different types of solid tumors are named for the type of cells thatform them (such as sarcomas, carcinomas, and lymphomas). Examples ofsolid tumors, such as sarcomas and carcinomas, include fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and othersarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreaticcancer, breast cancer, lung cancers, ovarian cancer, prostate cancer,hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma,adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma,papillary thyroid carcinoma, pheochromocytomas sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas, medullarycarcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bileduct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer,testicular tumor, seminoma, bladder carcinoma, melanoma, and CNS tumors(such as a glioma (such as brainstem glioma and mixed gliomas),glioblastoma (also known as glioblastoma multiforme) astrocytoma, CNSlymphoma, germinoma, medulloblastoma, Schwannoma craniopharyogioma,ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,oligodendroglioma, menangioma, neuroblastoma, retinoblastoma and brainmetastases).

The present invention also provides a medical device, including at leastone CAR protein, a polynucleotide sequence encoding a CAR, or a hostcell expressing a CAR, wherein the device is suitable for administeringthe at least one conditionally active CAR by at least one mode selectedfrom parenteral, subcutaneous, intramuscular, intravenous,intrarticular, intrabronchial, intraabdominal, intracapsular,intracartilaginous, intracavitary, intracelial, intracelebellar,intracerebroventricular, intracolic, intracervical, intragastric,intrahepatic, intramyocardial, intraosteal, intrapelvic,intrapericardiac, intraperitoneal, intrapleural, intraprostatic,intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal,intrasynovial, intrathoracic, intrauterine, intravesical, bolus,vaginal, rectal, buccal, sublingual, intranasal, or transdermal.

In a further aspect, the invention provides a kit including at least oneCAR protein, a polynucleotide sequence encoding a CAR, or a host cellexpressing a CAR, in lyophilized form in a first container, and anoptional second container including sterile water, sterile bufferedwater, or at least one preservative selected from the group consistingof phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol,phenylmercuric nitrite, phenoxyethanol, formaldehyde, chlorobutanol,magnesium chloride, alkylparaben, benzalkonium chloride, benzethoniumchloride, sodium dehydroacetate and thimerosal, or mixtures thereof inan aqueous diluent. In one aspect, in the kit, the concentration ofconditionally active CAR or a specified portion or variant in the firstcontainer is reconstituted to a concentration of about 0.1 mg/ml toabout 500 mg/ml with the contents of the second container. In anotheraspect, the second container further includes an isotonic agent. Inanother aspect, the second container further includes a physiologicallyacceptable buffer. In one aspect, the disclosure provides a method oftreating at least one wild-type protein mediated condition, includingadministering to a patient in need thereof a formulation provided in akit and reconstituted prior to administration.

Also provided is an article of manufacture for human pharmaceutical ordiagnostic use including a packaging material and a container includinga solution or a lyophilized form of at least one CAR protein,polynucleotide sequence encoding a CAR, or a host cell expression a CAR.The article of manufacture can optionally include having the containeras a component of a parenteral, subcutaneous, intramuscular,intravenous, intrarticular, intrabronchial, intraabdominal,intracapsular, intracartilaginous, intracavitary, intracelial,intracelebellar, intracerebroventricular, intracolic, intracervical,intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic,intrapericardiac, intraperitoneal, intrapleural, intraprostatic,intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal,intrasynovial, intrathoracic, intrauterine, intravesical, bolus,vaginal, rectal, buccal, sublingual, intranasal, or transdermal deliverydevice or system.

In some embodiments, the present invention provides a method includingretrieving cytotoxic cells from a subject, genetically engineering thecytotoxic cells by introducing a CAR gene of the present invention intothe cytotoxic cells, and administering the genetically engineeredcytotoxic cells to the subject. In some embodiments, the cytotoxic cellsare selected from T cells, naive T cells, memory T cells, effector Tcells, natural killer cells, and macrophages. In one embodiment, thecytotoxic cells are T cells.

In one embodiment, the T cells are obtained from a subject. T cells canbe obtained from a number of sources, including peripheral bloodmononuclear cells, bone marrow, lymph node tissue, cord blood, thymustissue, tissue from a site of infection, ascites, pleural effusion,spleen tissue, and tumors. In certain embodiments of the presentinvention, any number of T cell lines available in the art, may be used.In certain embodiments of the present invention, T cells can be obtainedfrom blood collected from a subject using any number of techniques knownto the skilled artisan, such as Ficoll™ separation.

In one preferred embodiment, cells from the circulating blood of anindividual are obtained by apheresis. The apheresis product typicallycontains lymphocytes, including T cells, monocytes, granulocytes, Bcells, other nucleated white blood cells, red blood cells, andplatelets. In one embodiment, the cells collected by apheresis may bewashed to remove the plasma fraction and to place the cells in anappropriate buffer or media for subsequent processing steps. In oneembodiment of the invention, the cells are washed with phosphatebuffered saline (PBS). In an alternative embodiment, the wash solutionlacks calcium and may lack magnesium or may lack many if not alldivalent cations. Again, surprisingly, initial activation steps in theabsence of calcium lead to magnified activation. As those of ordinaryskill in the art would readily appreciate a washing step may beaccomplished by methods known to those in the art, such as by using asemi-automated “flow-through” centrifuge (for example, the Cobe 2991cell processor, the Baxter CytoMate, or the Haemonetics Cell Saver 5)according to the manufacturer's instructions. After washing, the cellsmay be resuspended in a variety of biocompatible buffers, such as, forexample, Ca²⁺-free, Mg²⁺-free PBS, PlasmaLyte A, or another salinesolution with or without buffer. Alternatively, the undesirablecomponents of the apheresis sample may be removed and the cells directlyresuspended in culture media.

In another embodiment, T cells are isolated from peripheral blood bylysing the red blood cells and depleting the monocytes, for example, bycentrifugation through a PERCOLL™ gradient or by counter-flowcentrifugal elutriation. A specific subpopulation of T cells, such asCD3⁺, CD28⁺, CD4⁺, CD8⁺, CD45RA⁺, and CD45RO⁺ T cells, can be furtherisolated by positive or negative selection techniques. For example,enrichment of a T cell population by negative selection can beaccomplished with a combination of antibodies directed to surfacemarkers unique to the negatively selected cells. One method is cellsorting and/or selection via negative magnetic immunoadherence or flowcytometry that uses a cocktail of monoclonal antibodies directed to cellsurface markers present on the cells negatively selected. To enrich CD4⁺cells by negative selection, a monoclonal antibody cocktail typicallyincludes antibodies to CD 14, CD20, CD11b, CD 16, HLA-DR, and CD8. Incertain embodiments, it may be desirable to enrich for or positivelyselect for regulatory T cells which typically express CD4⁺, CD25⁺,CD62L^(hi), GITR⁺, and FoxP3⁺.

For example, in one embodiment, T cells are isolated by incubation withanti-CD3/anti-CD28 (i.e., 3×28)-conjugated beads, such as DYNABEADS®M-450 CD3/CD28 T, for a time period sufficient for positive selection ofthe desired T cells. In one embodiment, the time period is about 30minutes. In a further embodiment, the time period ranges from 30 minutesto 36 hours or longer and all integer values there between. In a furtherembodiment, the time period is at least 1, 2, 3, 4, 5, or 6 hours. Inyet another preferred embodiment, the time period is 10 to 24 hours. Inone preferred embodiment, the incubation time period is 24 hours. Forisolation of T cells from patients with leukemia, use of longerincubation times, such as 24 hours, can increase cell yield. Longerincubation times may be used to isolate T cells in any situation wherethere are few T cells as compared to other cell types, such in isolatingtumor infiltrating lymphocytes (TIL) from tumor tissue or fromimmune-compromised individuals. Further, use of longer incubation timescan increase the efficiency of capture of CD8+ T cells. Thus, by simplyshortening or lengthening the time T cells are allowed to bind to theCD3/CD28 beads and/or by increasing or decreasing the ratio of beads toT cells (as described further herein), subpopulations of T cells can bepreferentially selected for or against at culture initiation or at othertime points during the process. Additionally, by increasing ordecreasing the ratio of anti-CD3 and/or anti-CD28 antibodies on thebeads or other surface, subpopulations of T cells can be preferentiallyselected for or against at culture initiation or at other desired timepoints. The skilled person would recognize that multiple rounds ofselection can also be used in the context of this invention. In certainembodiments, it may be desirable to perform the selection procedure anduse the “unselected” cells in the activation and expansion process.“Unselected” cells can also be subjected to further rounds of selection.

The obtained cytotoxic cells are then genetically engineered asdescribed herein. A polynucleotide encoding the CAR, typically locatedin an expression vector, is introduced into the cytotoxic cells suchthat the cytotoxic cells will express, preferably stably, the CAR. Thepolynucleotide encoding the CAR is typically integrated into thecytotoxic cell host genome. In some embodiments, the polynucleotideintroduction need not result in integration but rather only transientmaintenance of the polynucleotide introduced may be sufficient. In thisway, one could have a short term effect, where cytotoxic cells could beintroduced into the host and then turned on after a predetermined time,for example, after the cells have been able to migrate to a particularsite for treatment.

Depending upon the nature of the cytotoxic cells and the diseases to betreated, the genetically engineered cytotoxic cells may be introducedinto the subject, e.g. a mammal, in a wide variety of ways. Thegenetically engineered cytotoxic cells may be introduced at the site ofthe tumor. In one embodiment, the genetically engineered cytotoxic cellsnavigate to the cancer or are modified to navigate to the cancer. Thenumber of genetically engineered cytotoxic cells that are employed willdepend upon a number of factors such as the circumstances, the purposefor the introduction, the lifetime of the cells, the protocol to beused. For example, the number of administrations, the ability of thecells to multiply, and the stability of the recombinant construct. Thegenetically engineered cytotoxic cells may be applied as a dispersioninjected at or near the site of interest. The cells may be in aphysiologically-acceptable medium.

It should be appreciated that the treatment method is subject to manyvariables, such as the cellular response to the CAR, the efficiency ofexpression of the CAR by the cytotoxic cells and, as appropriate, thelevel of secretion, the activity of the expressed CAR, the particularneed of the subject, which may vary with time and circumstances, therate of loss of the cellular activity as a result of loss of geneticallyengineered cytotoxic cells or the expression activity of individualcells, and the like. Therefore, it is expected that for each individualpatient, even if there were universal cells which could be administeredto the population at large, each patient would be monitored for theproper dosage for the individual, and such practices of monitoring apatient are routine in the art.

The following examples are illustrative, but not limiting, of themethods of the present disclosure. Other suitable modifications andadaptations of the variety of conditions and parameters normallyencountered in the field, and which are obvious to those skilled in theart, are within the scope of this disclosure.

EXAMPLES Example 1: Generation of scFv Conditionally Active Antibodies

Two conditionally active antibodies (CAB-scFv-63.9-4 andCAB-scFv-63.9-6) for a drug target antigen X1 were expressed ashomodimers with wild type human IgG1 Fc (resulting in bivalentantibodies CAB-scFv-63.9-4-01 and CAB-scFv-63.9-6-01 in FIGS. 2-3), aswell as heterodimers in the knob-in-hole system resulting in amonovalent scFv (resulting in monovalent antibodies scFvCAB-scFv-63.9-4-02 and CAB-scFv-63.9-6-02 in FIGS. 2-3).

The binding affinities of these antibodies to the drug target antigen X1at pH 6.0 and pH 7.4 were measured by the ELISA assay. As show in FIG.2, the scFv antibodies showed affinities to drug target antigen X1 atboth pH 6.0 and pH 7.4, which were comparable to the full bivalentantibodies. Further, the selectivity of these scFv antibodies at pH 6.0over pH 7.4 as shown in FIG. 3 was also comparable to the full bivalentantibodies. This example demonstrated that the conditionally activeantibodies of the present invention have comparable affinity andselectivity either as scFv antibodies or full bivalent antibodies. Thus,the conditionally active antibodies of the present invention may beinserted as a single DNA chain in a DNA molecule that encodes CAR in theCAR-T platform of the present invention.

Example 2: scFv Antibodies Against Target Antigen X1 for ConstructingCAR-T Cells

Conditionally active antibodies for the drug target antigen X1 weregenerated by simultaneously screening for selectivity and affinity, aswell as expression level at both pH 6.0 and pH 7.4, in accordance withone embodiment of the present invention. The screening was done in serumusing a FLAG tag because there were human antibodies in the serum whichmight cause false positives for the screening. The screening buffer wasa carbonate buffer (krebs buffer with ringer—standard buffer butdifferent from PBS). The generated conditionally active antibodies werefound to have a higher affinity to the drug target antigen X1 at pH 6.0but lower affinity to the same drug target antigen X1 at pH 7.4, both incomparison with the wild-type antibody. Further, these conditionallyactive antibodies all have high expression levels as shown in Table 2below, with column “Clone” showing the antibodies and the expressionlevel “mg/ml” being shown in the second column.

The clones of these antibodies were sent to a service provider with arequested expression level (“amount ordered”, expected expressionlevels). However, the actual expression levels of these antibodies(“amount delivered”) were very high and exceeded the expected expressionlevels.

TABLE 2 Conditionally active antibodies with high expression levelsamount amount Clone mg/ml targeted obtained BAP063.6-hum10F10-FLAG 7 150294 BAP063.6-HC-H100Y-FLAG 6.6 150 238 BAP063.8-LC046HC04-FLAG 7 200332.5 BAP063.8-LC062HC02-FLAG 5.8 200 220.4 BAP063.9-13-1-FLAG 5.3 50123 BAP063.9-29-2-FLAG 4.9 50 102 BAP063.9-45-2-FLAG 5.4 50 129BAP063.9-13-3-FLAG 5.9 50 130 BAP063.9-21-3-FLAG 5.3 50 117BAP063.9-21-4-FLAG 7 50 176 BAP063.9-29-4-FLAG 8.2 50 196BAP063.9-48-3-FLAG 7 50 125 BAP063.9-49-4-FLAG 5.3 50 126BAP063.9-61-1-FLAG 5.1 50 97 BAP063.9-61-2-FLAG 5 50 92

The conditionally active antibodies did not show aggregation in a bufferas demonstrated in FIG. 4, using BAP063.9-13-1 antibody as an example.The BAP063.9-13-1 antibody was analyzed by size exclusionchromatography. In FIG. 4, only one peak was detected, demonstratinglittle or no aggregation of the antibody.

The conditionally active antibodies were also assayed using surfaceplasmon resonance (SPR) to measure their on and off rates to the drugtarget antigen X1. The SPR assay has been known to measure on and offrates for the conditionally active antibodies. The SPR assay wasperformed in the presence of bicarbonate. The in vivo on and off rate(in animals and humans) of the conditionally active antibodies is a veryimportant feature for the conditionally active antibodies.

It was observed that the conditionally active antibodies have quickon-rates at pH 6.0 and slower on-rates at pH 7.4, in comparison with thenegative control (BAP063 10F10 which has similar on-rates at both pH 6.0and pH 7.4) (FIG. 5). In addition, raising the temperature from roomtemperature to 60° C. does not significantly alter the SPR assay results(FIG. 5). The SPR assay also showed that these conditionally activeantibodies were highly selective at pH 6.0 as compared to pH 7.4 (FIGS.6A-6B show one antibody as an example).

The conditionally active biological antibodies are summarized in Table3. Two of the antibodies were expressed as scFv (BAP063.9-13.3 andBAP063.9-48.3), which were ready to be inserted into a CAR in the CAR-Tplatform. Incubating the antibodies at 60° C. for one hour did notchange the affinities of most of the antibodies (“Thermostability”). Inthe two columns reporting data using SPR to measure binding activity atpH 6.0 and pH 7.4 (the last two columns of Table 3), a comparison wasmade to “BAP063.6-hum10F10-FLAG” (a negative control, second row inTable 3). The selectivity of these antibodies may be determined by thedifferences between the data in the two last columns. The two scFvantibodies had very high selectivity (75% and 50% at pH 6 over 0% at pH7.4).

TABLE 3 Summary of the conditionally active antibodies Increased bindingAggregation at pH 7.4 SPR SPR CAB amount amount (PBS, Thermostabilityafter heat KD [M] activity activity Clone scFv mg/ml ordered deliveredpH 7.4) (1 h 60° C.) treatment Ka [M · s] Kd [s^(−1]) pH 6.0 pH 6.0 pH7.4 BAP063.6- 7 150 294 No 100% No 5.14E+06 8.38E−04 1.63E−10 100% 100%hum10F10- FLAG BAP063.6- 6.6 150 238 N.D. 2.41E+06 5.12E−03 2.12E−09 80%40% HC-H100Y- FLAG BAP063.9- 5.3 50 123 No 100% Yes 1.98E+06 2.88E−031.46E−09 100% 75% 13-1-FLAG BAP063.9- 4.9 50 102 No 100% Yes 1.19E+062.14E−03 1.79E−09 90% 50% 29-2-FLAG BAP063.9- 5.4 50 129 No reduced Yes1.53E+06 2.31E−03 1.51E−09 75% 25% 45-2-FLAG BAP063.9- Yes 5.9 50 130 No100% Yes 1.42E+06 1.82E−03 1.28E−09 75% 0% 13-3-FLAG BAP063.9- 5.3 50117 No 100% No 1.53E+06 4.13E−03 2.69E−09 50% 25% 21-3-FLAG BAP063.9- 750 176 No 100% No 1.03E+06 3.26E−03 3.16E−09 50% 0% 21-4-FLAG BAP063.9-8.2 50 196 No 100% (Yes) 1.40E+06 2.21E−03 1.58E−09 75% 0% 29-4-FLAGBAP063.9- Yes 7 50 125 <5% reduced No 8.92E+05 2.33E−03 2.61E−09 50% 0%48-3-FLAGComparative Example A: CAR-T Cells with Non-Conditionally ActiveAntibody Against Target Antigen X1

A non-conditionally active scFv antibody against target antigen X1 wasused to construct CAR-T cells that bind to target antigen X1 or CHOcells expressing target antigen X1 on the cell surface (CHO-X1), FIGS.7A-7B. The non-conditionally active antibody was used as the ASTR of theCAR molecule that was inserted into T cells to construct CAR-T cellsthat can bind to target antigen X1.

As a comparison, CHO cells that do not express target antigen X1 weretreated with: (1) T cells not transduced with a CAR molecule, (2) Tcells transduced with a CAR molecule that does not bind to targetantigen X1, and (3) T cells transduced with a CAR molecule with anon-conditionally active antibody against target antigen X1 (FIG. 7A).The CHO cell population is indicated by the cell index (Y-axis in FIG.7A), with a decrease in cell index indicating cytotoxicity (cellkilling) by the CAR-T cells.

Referring to FIG. 7A, before addition of the T cells, the CHO cellsshowed growth. After addition of the CAR-T cells that bind to targetantigen X1, the cell index initially decreased, indicating non-specificcytotoxicity of the T cells. However, the CHO cells resumed growingshortly thereafter. More importantly, the differences among the threetreatments were insignificant, indicating no significant cytotoxicity tothe CHO cells that do not express target antigen X1 of the CAR-T cellswith the non-conditionally active antibody against target antigen X1.

CHO cells that express target antigen X1 were then treated in the samemanner as above with: (1) T cells not transduced with a CAR molecule,(2) T cells transduced with a CAR molecule that does not bind to targetantigen X1, and (3) T cells transduced with a CAR molecule with anon-conditionally active antibody against target antigen X1 (FIG. 7B).After addition of the T cells, the cell index is significantly decreasedby the treatment with the CAR-T cells with the non-conditionally activeantibody against target antigen X1, but not by the other two treatments,indicating cytotoxicity to the CHO-X1 cells that express target antigenX1 by the CAR-T cells with the non-conditionally active antibody againsttarget antigen X1.

Example 3: CAR-T Cells with a Conditionally Active scFv Antibody AgainstTarget Antigen X1

A conditionally active scFv antibody against target antigen X1 was usedto construct a CAR molecule. T cells were transduced with the CARmolecule such that the T cells expressed the CAR molecule (CAR-T cells).CHO cells expressing target antigen X1 (CHO-63 cells) or regular CHOcells that do not express target antigen X1 (CHO cells) were separatelytreated with the CAR-T cells. Non-transduced T-cells (without the CARmolecule) were used as a control (FIGS. 8A-8B).

Referring to FIG. 8A, CHO cells that do not express the target antigenX1 were treated with the CAR-T cells and non-transduced T-cells. Therewas no significant difference between the two treatments, indicating nocytotoxicity of the CAR-T cells to the CHO cells. Referring to FIG. 8Bwhere CHO cells expressing target antigen X1 (CHO-63) were similarlytreated, the CAR-T cells with a conditionally active antibody againsttarget antigen X1 significantly reduced the CHO-63 cell population, incomparison with non-transduced T-cells. This indicated that CAR-T cellswith a conditionally active antibody against target antigen X1 werecytotoxic to the CHO-63 cells.

The CAR-T cells, once bound to target antigen X1, induced cytotoxicity.This effect was confirmed by measurement of the levels of the cytokinesinterferon gamma (INFg) and IL2. The cytokine data is shown in FIGS.9A-9B. In FIG. 9A, the binding of CAR-T cells with target antigen X1 onthe CHO-63 cells triggered significant release of INFg, in comparisonwith non-transduced T cells, as shown by the increased cytokine levelsthat were observed. Similarly, in FIG. 9B, the binding of CAR-T cellswith target antigen X1 on the CHO-63 cells triggered significant releaseof IL2, in comparison with non-transduced T cells, as shown by theincreased cytokine levels that were observed.

Example 4: CAR-T Cells with a Conditionally Active scFv Antibody AgainstTarget Antigen X2

Conditionally active scFv antibodies against target antigen X2 wereproduced. Their binding activity to target antigen X2 was measured usingan ELISA essay (FIG. 10).

One of the scFv antibodies shown in FIG. 10, scFv-116101, was used toconstruct CAR molecules for producing CAR-T cells (116101 CAR-T). Theconstructed CAR-T cells were used to target Daudi cells that expresstarget antigen X2. The negative controls were T-cells not transducedwith a CAR molecule (non-transduced T cells) and CAR-T cells transducedwith a CAR molecule not capable of binding to target antigen X2 (non-X2scFv CAR-T). The results are shown in FIG. 11A. The ratio of the numberof T cells to the number of Daudi cells in these treatments was 10:1.The CAR-T cells with the scFv antibody targeting target antigen X2 onthe Daudi cells (116101 CAR-T) induced significant cell death for theDaudi cells as shown by the higher dead/live cell ratio in FIG. 11A.

HEK293 cells were treated with the same T cells as were used to treatthe Daudi cells. The results are shown in FIG. 11B. Since HEK293 cellsdo not express target antigen X2 on the cell surface, the CAR-T cellswith the scFv antibody targeting target antigen X2 (116101 CAR-T) didnot induce significant cell death in the HEK293 cells, as compared withthe negative controls (FIG. 11B).

Example 5: Cytokine Release of CAR-T Cells with Antibodies AgainstTarget Antigens X1 and X2

The cytokine release induced by binding of CAR-T cells with targetantigens was measured in this example. FIGS. 12A-12B show INFg and IL2release after binding of CAR-T cells containing a conditionally activescFv antibody against target antigen X1 with CHO-63 cells expressingtarget antigen X1. After treating the CAR-T cells with these CHO-63cells for 24 hours, there was a significant increase both the INFg andIL2 cytokine levels indicating release of both INFg and IL2 cytokines,in comparison with controls where the same CAR-T cells were used totreat CHO cells that do not express target antigen X1. Further, T cellsnot transduced with a CAR molecule and CAR-T cells that did not bindwith target antigen X1 did not result in significant release of INFg andIL2 cytokines.

FIGS. 13A-13B show INFg and IL cytokine levels after binding of CAR-Tcells containing a conditionally active scFv antibody against targetantigen X2 with Rajib cells and Daudi cells both of which express targetantigen X2. After treating the Rajib cells and Daudi cells with CAR-Tcells for 24 hours, a significant increase in INFg and IL2 cytokinelevels was observed, in comparison with controls where the same CAR-Tcells were used to treat HEK293 cells that do not express target antigenX2. Further, T cells not transduced with a CAR molecule and CAR-T cellsthat did not bind with target antigen X2 did not significantly increasecytokine levels thereby indicating a failure to induce significantrelease of INFg and IL2 cytokines.

Example 6: Conditionally Active scFv Antibodies Against Target AntigenX3

Five conditionally active scFv antibodies against target antigen X3 wereselected. The selected conditionally active scFv antibodies are moreactive at pH 6.0 than at pH 7.4. These conditionally active scFvantibodies may be used to construct CAR-T cells binding to cellsexpressing target antigen X3.

It is to be understood, however, that even though numerouscharacteristics and advantages of the present invention have been setforth in the foregoing description, together with details of thestructure and function of the invention, the disclosure is illustrativeonly, and changes may be made in detail, especially in matters of shape,size and arrangement of parts within the principles of the invention tothe full extent indicated by the broad general meanings of the terms inwhich the appended claims are expressed.

1-21. (canceled)
 22. A chimeric antigen receptor for binding with atarget antigen, comprising: i. at least one antigen specific targetingregion evolved from a parent protein or a domain thereof and having adecrease in activity in an assay at a normal physiological conditioncompared to a same activity in an assay under an aberrant conditioncompared to the antigen specific targeting region evolved from theparent protein or the domain thereof, the aberrant condition is acondition selected from conditions present in a tumor microenvironment,a brain extracellular fluid, a stem cell niche, a lymph node, a tonsil,an adenoid, a sinus, and a synovial fluid; ii. a transmembrane domain;and iii. an intracellular signaling domain.
 23. The chimeric antigenreceptor of claim 22, wherein the antigen specific targeting region hasa decrease in a binding affinity to the target antigen at the normalphysiological condition compared to the antigen specific targetingregion of the parent protein or the domain thereof. 24-25. (canceled)26. The chimeric antigen receptor of claim 22, further comprising anextracellular spacer domain or at least one co-stimulatory domain. 27.(canceled)
 28. The chimeric antigen receptor of claim 22, wherein the atleast one antigen specific targeting region is selected from anantibody, a ligand, a receptor binding domain of a ligand, a receptor, aligand binding domain of a receptor, an affibody, and combinationsthereof.
 29. The chimeric antigen receptor of claim 28, wherein the atleast one antigen specific targeting region is an antibody selected froma full length antibody, a single-chain antibody, an Fab fragment, anFab′ fragment, an (Fab′)2 fragment, an Fv fragment, and a divalentsingle chain antibody or a diabody.
 30. The chimeric antigen receptor ofclaim 26, wherein the extracellular spacer domain is selected from an Fcfragment of an antibody, a hinge region of an antibody, a CH2 region ofan antibody, a CH3 region of an antibody, an artificial spacer sequenceand combinations thereof.
 31. The chimeric antigen receptor of claim 22,wherein the transmembrane domain is selected from an artificialhydrophobic sequence and transmembrane domains of a Type I transmembraneprotein, an alpha, beta or zeta chain of a T cell receptor, CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80,CD86, CD134, CD137, CD154, and combinations thereof.
 32. The chimericantigen receptor of claim 22, wherein the co-stimulatory domain isselected from co-stimulatory domains of proteins in the TNFRsuperfamily, CD28, CD137, CD134, DaplO, CD27, CD2, CD5, ICAM-1, LFA-1,Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, ICOS LIGHT, NKG2C, B7-H3, andcombinations thereof.
 33. The chimeric antigen receptor of claim 22,wherein the intracellular signaling domain is selected from cytoplasmicsignaling domains of a human CD3 zeta chain, FcyRIII, FcsRI, acytoplasmic tail of a Fc receptor, an immunoreceptor tyrosine-basedactivation motif (ITAM) bearing cytoplasmic receptors, TCR zeta, FcRgamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a,CD79b, CD66d, and combinations thereof.
 34. (canceled)
 36. The chimericantigen receptor of claim 22, wherein the normal physiological conditionis selected from a temperature, pH, osmotic pressure, osmolality,oxidative stress, electrolyte concentration, a concentration of glucose,lactic acid, pyruvate, nutrient components, metabolites, oxygen,carbonate, phosphate, or carbon dioxide, cell types, and nutrientavailability.
 37. (canceled)
 38. An expression vector, comprising apolynucleotide sequence encoding the chimeric antigen receptor of claim22.
 39. The expression vector of claim 38, where the expression vectoris selected from lentivirus vectors, gamma retrovirus vectors, foamyvirus vectors, adeno associated virus vectors, adenovirus vectors, poxvirus vectors, herpes virus vectors, engineered hybrid viruses, andtransposon mediated vectors.
 40. A genetically engineered cytotoxiccell, comprising a polynucleotide sequence encoding the chimeric antigenreceptor of claim
 22. 41. The genetically engineered cytotoxic cell ofclaim 40, wherein the cytotoxic cell is a T cell.
 42. (canceled)
 43. Thegenetically engineered cytotoxic cell of claim 40, wherein the cytotoxiccell is selected from a natural killer cell, an activated NK cells, aneutrophil, an eosinophil, a basophil, a B-cell, a macrophage and alymphokine-activated killer cell. 44-46. (canceled)
 47. A chimericantigen receptor for binding with a target antigen, comprising: i. atleast one antigen specific targeting region binding with the targetantigen; ii. one component selected from: (a) another antigen specifictargeting region connected to the at least one antigen specifictargeting region by a conditional linker having a first conformation atan aberrant condition for the at least two antigen specific targetingregions to bind to the target antigen at a higher binding activity thanin a second conformation of the conditional linker at a normalphysiological condition, (b) an extracellular spacer domain having afirst conformation at the aberrant condition for the at least oneantigen specific targeting region to bind to the target antigen at ahigher binding activity than a second conformation of the extracellularspacer domain at the normal physiological condition, and (c) anextracellular spacer domain having an enhanced ubiquitylation-resistanceat the aberrant condition as compared to the ubiquitylation-resistanceof the extracellular spacer domain at the normal physiologicalcondition; iii. a transmembrane domain; and iv. an intracellularsignaling domain.
 48. The chimeric antigen receptor of claim 47, whereinthe at least one antigen specific targeting region has a decrease inactivity in an assay at the normal physiological condition compared to asame activity in an assay under the aberrant condition. 49-52.(canceled)
 53. A method for treating a cancer in a subject, comprisingthe steps of: a. introducing an expression vector comprising apolynucleotide sequence encoding the chimeric antigen receptor of claim22 into a cytotoxic cell obtained from the subject to produce agenetically engineered cytotoxic cell; and b. administering thegenetically engineered cytotoxic cell to the subject.
 54. The method ofclaim 53, wherein the expression vector is selected from lentivirusvectors, gamma retrovirus vectors, foamy virus vectors, adeno associatedvirus vectors, adenovirus vectors, pox virus vectors, herpes virusvectors, engineered hybrid viruses, and transposon mediated vectors. 55.The method of claim 53, wherein the cytotoxic cell is a T cell. 56-61.(canceled)